ExpandA Huskie Line bus drives by part of the new Huskie mural in this Shaw Local file photo on Monday
on the Annie Glidden Road railroad underpass
DeKALB – Route changes could be coming to DeKalb’s public transit operations
with new bus schedules going into effect next month if approved
Northern Illinois University student weekend traffic
The estimated annual cost to make changes to seven bus routes would cost the city about $50,909, according to a proposal Neuenkirchen sent the city manager’s office on Feb. 19.
Neuenkirchen has proposed the city’s buses at Routes 10 and 11 stop making a U-turn at Twombly Road and Rosenow Way.
“The staff feel that this is an unsafe action,” Neuenkirchen said in his proposal. If approved, the changes would see the Routes 10 and 11 buses turn right onto Eden’s Gate Drive, take a left at the stop sign onto Adam’s Way, a left at the stop sign to turn onto Rosenow Way, and a left onto Twombly Road, which would service stops as the bus heads toward Annie Glidden Road.
“Transit staff have observed a need for additional transit access to Northwestern Medicine Immediate Care Sycamore,” 1850 Gateway Drive, according to Neuenkirchen’s proposal. That clinic is only serviced currently by Route 21, which is the only route that goes to Sycamore. This proposal would revise Route 18’s schedule to add a bus stop on Doser Drive at the immediate care facility from Ollie’s Custard, documents show. The plan would also a second transfer for riders between Routes 18 and 21.
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The cis-regulatory elements encoded in an mRNA determine its stability and translational output
While there has been a considerable effort to understand the factors driving mRNA stability
the regulatory frameworks governing translational control remain more elusive
We have developed a novel massively parallel reporter assay (MPRA) to measure mRNA translation
named Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP)
NaP-TRAP measures translation in a frame-specific manner through the immunocapture of epitope tagged nascent peptides of reporter mRNAs
We benchmark NaP-TRAP to polysome profiling and use it to quantify Kozak strength and the regulatory landscapes of 5’ UTRs in the developing zebrafish embryo and in human cells
Through this approach we identified general and developmentally dynamic cis-regulatory elements
as well as potential trans-acting proteins
We find that U-rich motifs are general enhancers
and upstream ORFs and GC-rich motifs are global repressors of translation
We also observe a translational switch during the maternal-to-zygotic transition
where C-rich motifs shift from repressors to prominent activators of translation
we show that microRNA sites in the 5’ UTR repress translation following the zygotic expression of miR-430
Together these results demonstrate that NaP-TRAP is a versatile
and powerful method to decode the regulatory functions of UTRs across different systems
measurements of translation efficiency often reflect the amalgamation of several isoforms of a given gene
each with a unique set of regulatory elements
This process is complicated further by the fact that endogenous transcripts contain multiple regulatory elements that exert differential and often competing effects on translation
In MPRAs where reporters are encoded in DNA
measurements of translation can be confounded by additional layers of transcriptional regulation
which quantify the number of ribosomes on each transcript through sucrose gradient fraction and immunocapture of epitope-tagged ribosomes
or ribosomes translating ORFs outside of the coding sequence may skew translation measurements
the methodological complexity of these assays has reduced the use of translation-based MPRAs across diverse systems
we develop NaP-TRAP (Nascent Peptide Translating Ribosome Affinity Purification)
a novel method to measure in-frame translation through the immunocapture of nascent peptides
by including an N-terminal FLAG tag in the coding sequences of reporter mRNAs
we enrich for reporters in a manner proportional to the number of active ribosomes translating the main ORF in frame
we benchmark NaP-TRAP to polysome profiling and use reporter assays of increasing complexity to validate this method
using NaP-TRAP we quantify the Kozak strength in zebrafish by measuring the translation of thousands of reporters simultaneously
we assess the regulatory potential of endogenous 5’ UTR sequences in the developing zebrafish embryo and HEK293T cells
we identify common and developmentally modulated motifs that regulate translation in vertebrates as well as potential effector RBPs known to bind these sequences
In doing so we demonstrate that NaP-TRAP is an accessible
which has the capacity to measure translation control across multiple systems and identify regulatory sequences in vivo
a Schematic detailing the NaP-TRAP method
FLAG-tagged nascent chain complexes of reporter mRNAs are enriched via an anti-FLAG immunoprecipitation
Translation is measured as a ratio of reporter reads in the pulldown relative to the input
b NaP-TRAP derived translation at 6 hpf (left
N = 3 replicates; 25 embryos per replicate) and fluorescence measurements at 24 hpf (right
+MO N = 9 embryos) in the presence (+ MO) or absence (- MO) of a translation blocking morpholino (two-sided unpaired t-test: ** p < 0.005
p = 0.0036; Fluorescence +/− MO p < 10−4; error bars = SEM)
c NaP-TRAP derived translation values for 3xFLAG-GFP-3xmiR-430 (blue) and 3xFLAG-GFP-3xmiR-204 (gray) at 2 hpf (N = 4 replicates; 25 embryos per replicate) and 4.3 hpf (N = 4 replicates; 25 embryos per replicate) (two-sided unpaired t-test: ** p < 0.01
p = 0.0071; miR-430 2hpf vs 4 hpf = 0.0001; error bars = SD)
Schematic representation of repression of translation exercised by miR-430
d NaP-TRAP derived translation values at 2 hpf (left
N = 3 replicates; 25 embryos per replicate) and quantification of band intensity of western blot and example immunoblot (right
N = 3 replicates; 5 embryos per replicate) of GFP reporters with poly-A tails of 0
60 and 90 As in the presence of a DsRED injection control
NaP-TRAP derived translation values are strongly correlated with mean ribosome load (polysome profiling) for a 5’ UTR mRNA reporter library (two-sided Pearson’s R = 0.92
f A cumulative density plot of residuals (NaP-TRAP translation – predicted translation) for reporters containing oORFs (N = 247 reporters)
and no upstream start codons (N = 1993 reporters) (p-values were calculated using a two-sided Mann-Whitney U-test; oORF vs noAUG p < 10−52; oORF vs uORF p < 10−29; uORF vs noAUG p < 10−60)
Together these results demonstrate that: (1) NaP-TRAP measures the translation of individual reporters targeting general and developmentally dynamic cis-regulatory elements and (2) NaP-TRAP derived translation values are quantitative and correlate with protein output
these experiments highlight the versatility of the method as they demonstrate the capacity of NaP-TRAP to quantify cis-regulation mediated by the 5’ and 3’ UTRs
Taken together these results demonstrate that: (1) NaP-TRAP is a quantitative MPRA method and (2) the frame specificity of NaP-TRAP derived translation values results in a more accurate measurement of protein output than MRL for reporters with out-of-frame translation
The translation of seven reporters with a Kozak score between 295-305 was measured using a dual luciferase-based assay (f)
Plot comparing relative luciferase activity (Nano luciferase / Firefly luciferase)
and NaP-TRAP translation values (two-sided Pearson’s R; N = 3 replicates; 5 embryos per replicate
This result not only highlights the importance of measuring Kozak strength experimentally
but also demonstrates that NaP-TRAP derived translation values correlate well with protein production
these results demonstrate that NaP-TRAP can be employed as a MPRA
By measuring the translation of thousands of reporters simultaneously
we quantify and model Kozak strength in a vertebrate system
Altogether these results demonstrate the following: (1) uAUGs are the most prominent repressive elements during early embryogenesis
and (2) 5’ UTR regulatory landscapes are dynamic during development
a The translation of the 5’ UTR library at 2 hpf and 6 hpf using NaP-TRAP (two-sided Pearson’s R; N = 8,529 reporters)
Reporters were divided into four groups based on their translation rank at both time-points: repressed
b–i Fold-change enrichment and depletion for all pentamers in each group from (a) relative to the reporter library (one-sided hypergeometric test to calculate significance; Bonferroni corrected p-value threshold
Using hierarchical clustering sequence motifs were generated from enriched pentamers
The cumulative distribution of translation for reporters enriched (red
bottom 20%) in a representative motif were plotted (middle 80% gray)
The significance in the difference of the distributions of translation was determined using a two-sided Mann-Whitney U-test ((c)
j Schematic detailing the library of all possible tetramer repeats separated by dinucleotide spacers
k Translation measurements of the validation library at 2 and 6 hpf (two-sided Pearson’s R; N = 195 reporters)
Tetrameric reporters were labeled based on whether their repeat was enriched in the reporter groups described above (a)
l Heat map showing the significance of the overlap between eCLIP motifs for RBPs expressed in the early embryo (top 10) and the motifs enriched in each class in panel (a) (one-sided hypergeometric test)
m Cumulative distribution plot comparing the translation (2hpf / 6 hpf) of validation reporters identified as active post ZGA (pink) and repressed post ZGA (green) (two-sided Mann-Whitney U-test: p < 10−5)
motifs were generated from the reporter groups described above (a)
The information content of representative motifs was plotted: repressed (b)
Displayed E-values reflect the output of STREME
Tomtom was utilized to compare motifs to a database of human RBP motifs (E-value < 0.05)
these results identify sequence motifs in 5’UTRs that modulate general and dynamic translation during embryonic development
These results reveal 5’ UTR elements with differential regulatory activity on translation during early embryogenesis and identify potential trans-factors participating in this dynamic regulation
a Cumulative distributions of translation (6 hpf / 2 hpf) for reporters containing miR-430 (GCACUUA
AGCACUU; N = 212 reporters) or miR-1 (ACAUUCC
Reporters labeled in gray contain neither seed sequence (N = 8,242 reporters) (two-sided Mann-Whitney U test
p < 10−27 miR-430 reporters vs reporters with no seed; two-sided Mann-Whitney U test
p < 0.001 miR-1 reporters vs reporters with no seed)
c The effect of the number of complementary bases to miR-430 ((b)
N = 185 reporters) on the translation of reporters containing seed sequences at 6 hpf versus 2 hpf (two-sided Pearson’s R; (b) p < 10−3
d–e The effect of the number of complementary bases to miR-430 ((d)
N = 425 reporters) on translation for reporters containing seeds with a single mismatch at 6 hpf versus 2 hpf (two-sided Pearson’s R; (d) p < 0.8
g Plots showing the delta translation (Translation 6 hpf / 2hpf) of reporter fragments that tile the zebrafish 5’ UTR (ENSDART00000135384 / usp9 (f)
The seed sites of miR-430 (AGCAUUU) are labeled in green
i Schematic detailing dual luciferase assay measuring the inhibitory effect of miR-430 binding sites in the 5’ UTR (h)
4xmiR-430-nanoluc and 4xmiR-430-shuffled-nanoluc were injected in wild-type and miR-430 knockout embryos
Relative Luciferase Activity (RLU) values were normalized to each reporter (N = 3 replicates; 5 embryos per replicate; two-sided unpaired t-test; (i) p < 0.001; error bars = SD)
suggesting that microRNA complementarity is correlated with translation repression and that the seed is crucial for this interaction
Taken together these findings demonstrate that the zygotic expression of miR-430 represses the translation of mRNAs with 5’ UTR seed sites and suggest that miRNA target sites in the 5’ UTR can provide significant translational repression in vivo
c–f The translation measurements of four wild-type (wt) and four mutant (mut) reporters using NaP-TRAP at 2 hpf and 6 hpf
U’s in two active (U-rich) reporters were mutated to C’s (U > C)
whereas C’s in two active-post ZGA (C-rich) reporters were mutated to U’s (C > U
whereas mut reporters are labeled red (N = 3 replicates; 25 embryos per replicate; two-sided unpaired t-test
a Schematic detailing NaP-TRAP in HEK293T cells
Cells were transfected with the 5’ UTR library using lipid nanoparticles
b–d The distribution of translation values in HEK293T cells
The top and bottom 10% of reporters based on their translation values are labeled in orange and blue (N = 7506 reporters) (b)
A differential enrichment analysis identified pentamers enriched and depleted in repressed (c) and active (d) reporters
respectively (one-sided hypergeometric test with a Bonferroni corrected p-value
e Venn-diagram comparing the pentamers enriched in active reporters in HEK293T cells and zebrafish embryos at 2 and 6 hpf
f Translation values at 2 hpf in zebrafish compared to translation values in HEK293T cells (two-sided Pearson’s R)
Reporters were divided into four groups based on their translation rank at each condition: active (blue)
active in zebrafish at 2 hpf (pink) and active in HEK293T cells (green) (N = 7506 reporters)
g–n Fold-change enrichment and depletion for all pentamers in each group from (f) relative to the reporter library (one-sided hypergeometric test to calculate significance; Bonferroni corrected p-value threshold
bottom 20%) in representative motifs were plotted (middle 80% gray)
The significance in the difference of the distributions of translation was determined using a two-sided Mann-Whitney U-test ((h) p < 10−300
o Using STREME motifs were generated from the reporter groups described above (f)
The information content of representative motifs was plotted: repressed (g)
active in the developing zebrafish embryo at 2 hpf (k)
Displayed E-values reflected the output of STREME
Tomtom was utilized to compare motifs to a database of human RBP motifs
RBPs were displayed above their corresponding motif (E-value < 0.05)
and demonstrate its capacity to measure translation quantitatively
We use NaP-TRAP to characterize cis-regulatory elements in the 5’ and 3’ UTRs
and the regulatory landscape of the 5’ UTRs of mRNAs in developing zebrafish embryos and HEK293T cells
Using NaP-TRAP we have identified general and developmentally dynamic cis-regulatory elements
as well as characterized global changes to translation associated with early embryogenesis
by injecting or transfecting in vitro transcribed mRNA reporter libraries
NaP-TRAP eliminates the confounding effects of transcriptional regulation associated with MPRAs that introduce reporters as DNA
When quantifying the regulatory potential of the 5’ UTR
transcriptional bias may be more pronounced given the region’s proximity to the promoter sequence
We attribute this difference to the fact that NaP-TRAP measures frame-specific translation as only the nascent chains of ribosomes translating the main ORF are epitope-tagged and thereby immunocaptured
whereas in polysome profiling and other TRAP methods
inactive ribosomes or ribosomes translating outside of the main open reading frame may affect the measurements of translation
This prediction will improve our annotation of Kozak strength in the zebrafish
as well as our capacity to modulate protein output
Prior to gastrulation the pool of PAPBC1 (poly(A) binding protein cytoplasmic 1) is limited
driving competition between transcripts for PAPBC1
This competition is eliminated following gastrulation as the relative abundance of PAPBC1 and active ribosomes increases following zygotic genome activation
resulting in the decoupling of poly-A tail length and translation efficiency
While we do not know if EIF4G recruitment modulates translation activation in our experiments
the fact that U repeats are activators of translation across timepoints and experimental systems
suggests that this observation may be driven by a component of the canonical translation initiation machinery
suggest that the human translation machinery is equipped to overcome repressive G-rich and structural elements in zebrafish 5’ UTRs
Given the well-characterized effect of temperature on RNA structure
it is an intriguing possibility that these differences in translation regulation of G-rich elements may be related to differences in physiological body temperature between zebrafish and humans (28 C and 37 C
Future work could explore this phenomenon by measuring the translation of 5’ UTRs of multiple species across model systems and temperature ranges
and quantitative method that measures the translation of thousands of reporters simultaneously through the immunocapture of FLAG-tagged nascent peptides
b Through the over-expression of an HA-tagged RBP
NaP-TRAP can be employed in conjunction with an RNA immunoprecipitation experiment to measure the effect of RBP recruitment on translation
c–f NaP-TRAP quantifies translation in a frame-specific manner
Through the incorporation of additional epitope tags in frames 2,3 or ORFs outside of the main open reading frame
the NaP-TRAP method can be utilized to: (1) measure out-of-frame translation in the main ORF (c)
(2) detect IRES sequences in an unbiased manner through the use of a bicistronic reporter (d)
and (4) quantify stop codon readthrough (f)
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact
Plasmids generated in this study are available from the Lead Contact on request
Wild-type zebrafish embryos were obtained through natural mating of TU-AB strain of mixed ages (5-18 months)
Mating pairs were randomly chosen from a pool of 60 males and 60 females allocated for each day of the month
Fish lines were maintained following the International Association for Assessment and Accreditation of Laboratory Animal Care research guidelines and approved by the Yale University Institutional Animal Care and Use Committee (IACUC)
a 3xFLAG tag was incorporated after the first 18 nucleotides of GFP-3xAID* (auxin-inducible domain) using an In-Fusion® HD Cloning kit (Takara #638946) (F: 3xFLAG_inf_fwd; R: AID_inf_rev)
While AID* domains were included in the initial NaP-TRAP vector to enable future use of an auxin-inducible degron system
this system was not utilized in this study
The vector also included an SP6 promoter sequence and SV40 poly-adenylation signal
NaP-TRAP reporter and dsRED control (plasmid pCS2 + - DsRED) mRNAs were generated using a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340) from linearized reporter plasmids via NotI-HF® (NEB #R3189L) restriction enzyme digest
In vitro transcribed mRNAs were purified using a Monarch® RNA Cleanup Kit (NEB #T2040L) prior to injection
to assess the capacity of NaP-TRAP to measure microRNA-mediated repression in the 3’ UTR
two additional reporters were generated: (1) 3xFLAG-GFP-3xmiR-430 and (2) 3xFLAG-GFP-3xmiR-204
Three binding sites of either miR-430 or miR-204 were cloned into the 3’ UTR of 3x-FLAG-GFP-3xAID*
using an In-Fusion® HD Cloning kit (Takara #638946) (F: FGFP_inf_fwd; R: 3xmir430_inf_rev and 3xmir204_inf_rev
Single-cell embryos were injected with 20 pg of both the 3xFLAG-GFP-3xmiR-430 and 3xFLAG-GFP-3xmiR-204 mRNAs
Twenty-five embryos per replicate were collected and flash-frozen in liquid nitrogen at 2 and 4.3 hpf
To test if NaP-TRAP is able to capture the translational regulatory activity provided by poly(A) tail length in the early embryos
NaP-TRAP experiments with reporters harboring differential poly(A) tail length were performed
The 3xFLAG-GFP vector was amplified using a forward primer against SP6 and reverse primer against the SV40 poly(A) signal containing the differential number of untemplated Ts at the 5’-end (0
The amplicons were generated by performing a PCR (SP6_F and either 0 A/30 A/60 A/90A_R primers) using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 18 cycles at 98 °C
The amplicons were gel purified using Zymo Gel DNA recovery (Zymo
D4008) and recovered with DNA clean and concentrator kit (Zymo
PCR products were used for in vitro mRNA synthesis using mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340) generating NaP-TRAP reporters with various poly(A) tail length
the embryos were dechorinated using Pronase (Sigma
P8811-1G) and 1-cell stage embryos were injected with a mix of 40 pg of NaP-TRAP reporter with a specific poly(A) tail length and 40 pg of DsRed control
Twenty-five embryos per replicate were collected and flash-frozen in liquid nitrogen at 2 hpf for a total of three replicates per condition
Translation was then measured by NaP-TRAP and qPCR as mentioned below
*For methods detailing NaP-TRAP and qPCR translation measurements see sections: (1) NaP-TRAP (Nascent Peptide Translating Ribosome Affinity Purification) and (2) NaP-TRAP qPCR analysis
30A_rev: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTGTTGTTAACTTGTTTATTGC
60A_rev: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTGTTGTTAACTTGTTTATTGC
90A_rev: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTTGTTGTTAACTTGTTTATTGC)
To eliminate excess cytoplasmic mature 3xFLAG-GFP protein that may otherwise compete with nascent peptide for immunocapture
a C-terminal PEST domain was incorporated into the NaP-TRAP reporter plasmid using an In-Fusion® HD Cloning Kit (Takara #638946)
The 3xFLAG-GFP vector was amplified from the 3xFLAG-GFP-3xAID* plasmid (F: GFP_dd1_inf_fwd
whereas the PEST domain insert was generated using PCR overlap extension (F: PEST_fwd
3x-FLAG-GFP-PEST is referred to as 3xFLAG-GFP in the text and figures of this manuscript unless stated otherwise
NaP-TRAP reporter libraries were constructed using three different PCR reactions:
the common coding sequence and 3’ UTR of the reporters were amplified from the 3xFLAG-GFP plasmid
using a forward primer targeting the N-terminus of GFP and a reverse primer targeting the 3’ end of the 3’ UTR (F: 3xFLAG_GFP_fwd
a forward primer targeting the sequence immediately downstream of the variable Kozak sequence was used (F: ntrapK_GPF_fwd)
All PCR amplicons were gel purified using a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
reporter libraries were amplified from 1 ng of single-stranded DNA oligo pools using KAPA 2X HiFi HotStart ReadyMix (Roche #7958935001) for 10-20 cycles of 98 °C
For initial Kozak library generation see Random Kozak Library section
Each product was PCR purified using a DNA Clean & Concentrator-5 (Zymo #D4014)
to generate a template for in vitro transcription
a PCR overlap extension was performed between the reporter library and the purified 3xFLAG-GFP-PEST amplicon using KAPA HiFi HotStart ReadyMix (Roche #7958935001) (0.1-1 ng of template DNA)
primers targeting the 5’ SP6 promoter sequence and the 3’ end of the 3xFLAG-GFP-PEST amplicons were added (F: SP6_II_adapt
sv40-R: sv40_rev) followed by an additional 20 cycles with the same conditions
reporter libraries contained a 60A tail (pA-R: pCS2_3utr_60A)
For libraries with an SV40 polyadenylation signal
a reverse primer targeting the 3’ end of the SV40 poly-adenylation signal was used for the amplification of 3xFLAG-GFP-PEST and the assembly of reporter library (R: SV40_rev)
templates for in vitro transcription were gel purified using a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
Reporter mRNAs were generated using a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340)
In vitro transcribed mRNAs were purified using a Monarch® RNA Cleanup Kit (NEB #T2040L)
All libraries were injected into single-cell zebrafish embryos at 20 pg per embryo
The random Kozak library consisted of an I5 Illumina adaptor (5’- CCCTACACGACGCTCTTCCGATCT-3’) followed by the 5’ UTR of Xenopus beta-globin
six upstream and one downstream of the start codon
The Kozak library was generated by performing a PCR overlap extension using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 10 cycles of 98 °C
and 30 seconds respectively (F: kozak_ntrap_fwd
A custom single-stranded DNA oligo pool consisting of 11,088 oligos was ordered from GenScript (12 K oligo pool)
Each 170 nt oligo contained an Illumina I5 (5’- CCCTACACGACGCTCTTCCGATCT-3’) adaptor sequence
and 22 nt region with homology to the Kozak sequence and N-terminus of 3xFLAG-GFP (5’-GTAAACATGGTGAGCAAGGGCG-3’)
The variable region of the 5’ UTR library was generated using a custom script
tiling the 5’ UTRs of 1,775 maternally supplied genes and six IRES sequences (human AQP4
EMCV and crTMV) in 124 nucleotide segments every 25 nucleotides
A custom single-stranded DNA oligo pool consisting of 256 oligos was ordered from Twist Bioscience as part of a 12 K oligo pool
The design of the common regions of library were identical to that of the zebrafish 5’ UTR library
The variable region (124 nucleotides) consisted of repeats of all possible tetramers
Each repeat occurred 21 times and was separated by a dinucleotide spacer
The dinucleotide spacers were repeated in a pattern across the variable region (TC
These dinucleotide spacers were selected to prevent the creation of unintended upstream ORFs
The NaP-TRAP multi-frame library consisted of an I5 Illumina adaptor (5’- CCCTACACGACGCTCTTCCGATCT-3’) followed by a 5’ UTR containing three potential ORFs in frames 1
and a CDS consisting of 3xFLAG-HA-MYC-GFP-Nano-Luciferase
consisting of different combinations of no ORFs
was assembled through PCR overlap extension
using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 10 cycles of 98 °C
The overlap product was purified using a DNA Clean & Concentrator-5 (Zymo #D4014)
MF_ORF_fwd – 5’- CCCTACACGACGCTCTTCCGATCTACAGCACGATSGGGGATCTTCAGCTTTMACTGTTCAAGACACTCGATCAT-3’
CTCCTCGCCCTTGCTGACSATGGTGGCGGCGTKAAAGAGCAATACCCCCSATCGACTTTGGATKATGTACAGACTTCSATGATCGAGTGTCTTGAACAGT-3’
3xFLAG-HA-MYC-GFP-Nano-Luciferase was synthesized as a gene fragment from Twist Bioscience
To measure translation in all frames simultaneously
stop codons were eliminated from frames 2 and 3 of the CDS
Epitope tags were designed as three repeats of FLAG-HA and MYC
Single nucleotide spacers were placed in between epitope each tag to modulate the frame each of the epitopes were expressed in
The fragments were assembled using PCR overlap extension
After which primers were added to amplify the assembled fragments (F:mf_gfp_fwd
R: pcr_II_pA_rev) for an addition 10 cycles using the same cycling parameters
The product was then gel purified with a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
the multi-frame 5’ UTR and 3xFLAG-HA-MYC-GFP-Nano-Luciferase gene fragments were assembled through PCR overlap
Forward and reverse primers were added to amplify the assembled reporter library for an additional 10 cycles to add an SP6 promoter sequence and a hard encoded 60 A tail respectively (F: SP6_II_adapt
Following the PCR the library was gel extracted using a Monarch® DNA Gel Extraction Kit (NEB #T1020L) and the reporter mRNAs were in vitro transcribed using a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340) following manufacturer’s protocol
Reporter mRNAs were purified using a Monarch® RNA Cleanup Kit (NEB #T2040L) and injected into single-cell zebrafish embryos at 20 pg per embryo
The design of the spike-in reporters was identical to that of the zebrafish 5’ UTR and tetramer validation libraries
Each spike-in reporter contained a 20-nucleotide identifier (see below)
NaP-TRAP spike-ins were generated by performing a PCR overlap extension using KAPA HiFi HotStart ReadyMix (Roche #7958935001) (F: ntrap_sp1_fwd
ntrap_sp5_fwd; R: ntrap_sp_rev) (1 µL of each primer at 100 µM) for 10 cycles of 98 °C
Amplicons were gel purified using a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
the purified amplicons were cloned into the 3xFLAG-GFP-PEST vector using In-Fusion® HD Cloning (Takara #638946) (vector F: ntrap_spike_inf_fwd
plasmids were amplified using KAPA HiFi HotStart ReadyMix (Roche #7958935001) (F: Sp6-Il-adapt
Products were then gel purified with a Monarch® DNA Gel Extraction Kit (NEB #T1020L) and then in vitro transcribed with a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340)
mRNAs were purified with a Monarch® RNA Cleanup Kit (NEB #T2040L) prior to use
Spike-ins were pooled and added at the RNA extraction step at varying amounts of 1
Following zebrafish mating embryos were dechorionated using Pronase (Sigma-Aldrich #10165921001)
The embryos were then injected with 20 pg of in vitro transcribed mRNA at the single-cell stage
embryos were incubated at 28 C for 2 to 6 hours post fertilization
Stage matched embryos (64-cell and shield respectively) were flash-frozen in liquid nitrogen
and the validation libraries 50 embryos were collected per replicate
Frozen embryos were lysed in 500 µL of lysis buffer (see below)
For NaP-TRAP HEK293T cells were plated on 6-well plates coated with poly-d-lysine 24 hours prior to transfection
In accordance with the manufactures protocol
the cells were transfected with 2 ug of reporter library per well the using Lipofectamine™ MessengerMAX™ (ThermoFisher Scientific # LMRNA008)
HEK293T cells were grown on 10 cm2 cell culture plates coated with poly-d-lysine (1-2 million cells per plate)
Given the larger number of cells per replicate
cells were transfected with 10 ug of reporter library per plate
the cells were washed with 1x DPBS and fed with fresh pre-warmed media
HEK293T cells were treated with cycloheximide at a final concentration of 100 ug/mL for 10 minutes at 37 C and washed on ice three times with 1 mL of ice-cold PBS (with 100 ug/mL cycloheximide) prior to lysis
500 uL of lysis buffer (see below) was added to each well/plate
HEK293T cells were mechanically scrapped using a cell scraper
20 pg of mRNA reporter library were injected into each embryo
Given that the mRNA reporters are ~1.2 kb long and the molecular weight of a single base is around 340 daltons
the molecular weight of each reporter mRNA is estimated to be ~408 kilodaltons
Injecting 20 pg of mRNA should result in roughly 29.5 million mRNAs or in the case of the zebrafish 5’ UTR library (11,088 reporters) 2662 mRNAs per reporter per embryo
Given that 50 embryos per replicate were collected
there is estimated to be roughly 133,118 copies of each reporter at the start of each experiment
In the zebrafish 5’ UTR library experiments
at 6 hpf over 75% of reporters passed the QC (100 unique reads in the input)
suggesting that injecting 20 pg per embryo provided sufficient coverage
see Methods S1- A Detailed Protocol for NaP-TRAP
the following buffers were prepared: 10x salt buffer (150 mM Tris 7.4
40 U/mL RNaseOUT™ Recombinant Ribonuclease Inhibitor (ThermoFisher Scientific #10777019)
1x cOmplete™ EDTA-free Protease Inhibitor (Roche #11873580001))
and NaP-TRAP wash buffer (lysis buffer + 400 mM NaCl)
Zebrafish embryo and HEK293T cell lysates were incubated for 10 minutes at 4 °C
The lysate was passed through a 25-guage needle (5-10 times)
the sample was centrifuged at 16,000 g for 5 minutes at 4 °C
The supernatant was transferred to a new Eppendorf tube and 2 µL of DNase I (NEB #M0303L) was added
the samples were diluted to 1 mL using additional lysis buffer and 75 µL of lysate was collected from each sample to serve as an input
To capture tagged nascent chains of reporter mRNAs
an immunoprecipitation using anti-FLAG magnetic beads was performed
Magnetic beads were purchased from three suppliers during the course of the study due to supply chain issues (ANTI-FLAG® M2 Magnetic Beads
Millipore® #M8823; Anti-Flag Magnetic Beads
BioTools LLC #B26102; Pierce™ Anti-DYKDDDDK Magnetic Agarose
For the zebrafish and human cell experiments 10 µL and 20 µL of beads (binding capacity of >0.8 mg of FLAG peptide / mL) were utilized per replicate
The magnetic beads were washed with 800 µL of bead wash buffer three times prior to being added to the lysis solution
Upon addition of the lysate sample to magnetic beads
tubes were placed on a rotator at 4 °C for 2 hrs
the beads were washed with 800 µL of NaP-TRAP wash buffer three times
The beads (pulldown) and the inputs were then resuspended in 1 mL of Trizol (Invitrogen #15596-018)
For the zebrafish 5’ UTR and tetramer validation libraries
RNA extractions were performed in accordance with the manufacture’s protocol
RNA pellets were resuspended in 11 µL of nuclease-free H2O
The protocol of multi-frame NaP-TRAP mirrors that of single-frame NaP-TRAP with the exception of the pulldown step
the cell lysate was divided into three equal parts for the immunoprecipitation of FLAG
Each fraction was diluted to 1 mL with lysis buffer
for each replicate 10 μl of Anti-FLAG (Pierce™ Anti-DYKDDDDK Magnetic Agarose
Anti-HA (Pierce™ Anti-HA Magnetic Beads # 88836)
and Anti-MYC (Pierce™ Anti-c-Myc Magnetic Beads #88842) beads were washed and then added to their respective fractions
As described above the beads were incubated with the lysate for 2 hours at 4 C prior to being washed three times with 800 μl of NaP-TRAP wash buffer
The beads (pulldown) and the inputs were then resuspended in 1 mL of Trizol (Invitrogen #15596-018) and spike in reporters were added
HEK293T cells were incubated with 100 μg/ml cycloheximide for 10 min at 37 °C to arrest translation
After washing with prechilled 1x DPBS supplemented with 100 μg/ml cycloheximide
cells were lysed in NaP-TRAP lysis buffer by manual scrapping using a cell scraper
Cell lysates for polysome profiling were prepared following the same protocol as done for NaP-TRAP until the immunocapture stage
1 ml of cell lysates were layered onto 10–60% sucrose gradients
in a thin-walled ultracentrifuge tube and centrifuged in a Beckman SW‐40Ti rotor at 155,000 g
Gradients were fractionated in a Teledyne ISCO fractionator using >60% sucrose chase solution
Absorbance was monitored at 254 nm to obtain the polysome profile and a total of twenty-seven 500 μl fractions were collected at a flow rate of 1 ml/min
Fractions corresponding to the single monosome or polysome peak were pooled postcollection prior to RNA extraction
RNA was extracted using TRIzol (Thermo Fisher Scientific) reagent
500 μl was added to each fraction and vortexed
100 µl of chloroform was added and the mixture was vortexed and then incubated for 2-3 min at room temperature
for 30 min and the aqueous phase was transferred to a fresh Eppendorf tube
250 μl of isopropanol and 1 μl of GlycoBlue was added and vortexed
After incubating at room temperature for 10 mins
the samples were centrifuged at 14,000 g for 30 mins
The RNA pellets were washed with 80% ethanol
dried and resuspended in 11 ul of nuclease-free water
For a detailed protocol see Methods S1- A Detailed Protocol for NaP-TRAP
Reverse transcription primers (4 µM total) targeting the N-terminus of 3xFLAG GFP were added to purified input and pulldown RNAs (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-CTAC-10N UMI-TAAC-6 nt sample barcode-GCGTCATGGTCTTTGTAGTCCTC-3’; see NaP-TRAP barcode RT primers in Supplementary Table 1)
These primers contained a 6 nucleotide sample barcode and a 10 nucleotide Unique Molecular Identifier (UMI) to allow for demultiplexing and read deduplication
as well as a 3’ I7 Illumina adaptor sequence
To increase library complexity the primer pairs were staggered by 1 nt (e.g.
Reverse transcription was performed using the Superscript III kit (Invitrogen #18080044) in accordance with manufacturer’s instructions
Reverse transcription reactions were performed at 55 °C
cDNA from replicates were pooled and purified by adding AMPure XP Reagent (Beckman Coulter #A63881) at 1.8x of the original sample volume
Illumina I5 and I7 forward and reverse primers containing a 10-nucleotide index (see below) were utilized to amplify cDNA libraries via PCR (Kappa Polymerase Master Mix)
To reduce the number of PCR duplicates 12-18 cycles were utilized
Amplicons were purified by adding AMPure XP Reagent (Beckman Coulter #A63881) at 0.9x the volume of the PCR reaction
Libraries were sequenced on Illumina NovaSeq 6000 platform
5’- AATGATACGGCGACCACCGAGATCTACAC-10 nt index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
5’- CAAGCAGAAGACGGCATACGAGAT-10 nt index-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
where the NaP-TRAP reporter was the target and DsRed the control
Translation was calculated as a ratio of fold enrichment in the pulldown relative to the input
Translation blocking morpholino (GFP_qpcr_fwd
MicroRNA seeds in 3’ UTR (3xmir-430_qpcr_fwd
To validate NaP-TRAP translation measurements
single-cell zebrafish embryos were co-injected with mRNAs encoding nano and firefly luciferase (0.5 pg of nano / 19.5 pg of firefly)
Embryos were collected at either 2 hpf or 6 hpf and frozen in liquid nitrogen (5 embryos per replicate)
Nano and firefly luciferase activities were measured using the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega #N1610)
Firefly luciferase activity was utilized to normalize nano luciferase measurement across reporters
Firefly luciferase was amplified from a pCS2 + -Fluc plasmid using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 20 cycles of 98 °C
Products were gel purified with a Monarch® DNA Gel Extraction Kit (NEB #T1020L) and then in vitro transcribed with a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340)
mRNAs were purified with a Monarch® RNA Cleanup Kit (NEB #T2040L) prior to injection
We substituted NanoLuc luciferase (amplified from a gBlocks™ Gene Fragment
Integrated DNA Technologies IDT) for GFP in the 3xFLAG-GFP-PEST vector using In-Fusion® HD Cloning (Takara #638946) (3xFLAG-PEST vector F: PEST_fwd
R: FLAG_rev_inf; NanoLuc insert F: FLAG-Nluc_inf_fwd
Note: The PEST domain was included in NanoLuc constructs to reduce the half-life of the NanoLuc protein and thereby improve the sensitivity of the assay
To generate NanoLuc reporters 3xFLAG-NanoLuc-PEST was amplified using KAPA HiFi HotStart ReadyMix (Roche #7958935001) (F: 3xFLAG_GFP_fwd R: pcr_II_pa_rev) and gel purified using a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
3xFLAG-NanoLuc (amplicon from the previous section) was amplified with seven different forward primers (ntrap_k1_fwd
and ntrap_k7_fwd) and a common reverse primer (pcr_II_pa_rev) using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 20 cycles of 98 °C
Products were gel purified using a Monarch® DNA Gel Extraction Kit (NEB #T1020L)
To add an SP6 promoter sequence and a 60 A hard-encoded tail
the purified product (1 ng) was amplified using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 20 cycles of 98 °C
the input of one of the replicates of the Kozak reporter K4 was below the read filter cutoff in the NaP-TRAP Kozak Library experiment
Full-length reporters were constructed by performing a PCR overlap extension of the 5’ UTR products with the NanoLuc amplicon described in the Cloning NanoLuc section using KAPA HiFi HotStart ReadyMix (Roche #7958935001)
primers targeting the SP6 promoter sequence (F: Sp6-Il-adapt)
and the 3’ end of the nanoLuc amplicon were added (R: pCS2_3utr_60A)
The PCR reaction was continued for an additional 20 cycles at the same cycle conditions
Products were gel purified with a Monarch® DNA Gel Extraction Kit (NEB #T1020L) and then in vitro transcribed with the mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340)
Polysome validation Supplementary Fig. 1 (R1 = ENSDART00000104444_9
Zebrafish 5’ UTR library validation reporters (R13: slc16a1_fwd / slc16a1_rev
WT-R: 4xmiR-430-rev; MUT-F: 4xmiR-430-MUT-fwd
C- and U-rich wild-type and mutant reporters were generated through PCR overlap extension (Primers used: igf1ra_wt_fwd
atp6v0_wt_rev; atp6v0_mut_F atp6v0_mut_R) using KAPA HiFi HotStart ReadyMix (Roche #7958935001) for 10 cycles of 98 °C
respectively (1 µL of each primer at 100 µM)
The extension products were then gel purified using a using Monarch® DNA Gel Extraction Kit (NEB #T1020L)
Extension products were cloned into the 3xFLAG-GFP-3xAID* vector using In-Fusion® HD Cloning (Takara #638946) (vector F: 3xFLAG_GFP_F R: I5_inf_rev)
Plasmids were digested with NotI-HF® (NEB #R3189L) and in vitro transcribed with a mMESSAGE mMACHINE™ SP6 transcription kit (Invitrogen™ #AM1340)
Barcodes identifying replicates and UMIs were extracted from read two
Using ReadKnead the 5’ common region of the Kozak reporters were removed
we extracted the Kozak sequence (NNNNNNATGN) and discarded sequences that did not include ATG in positions 7-10
We then utilized a custom script to eliminate PCR duplicates
UMIs were considered identical if they had a Hamming Distance less than 2
Reporters with indels in the Kozak sequence or reporters without an AUG in the appropriate position were eliminated
For the zebrafish 5’ UTR and validation libraries, reads were mapped to a library-specific index using Bowtie274
Reads for each experiment were normalized by dividing the read counts of each reporter by the sum of the total number of reads mapped to the spike-ins
In the 5’ UTR reporter library (pA and sv40 in zebrafish) spike-in #3 was eliminated from the analysis
because in some of the samples the read counts for spike-in #3 did not correlate with amount of spike-in added
read counts were normalized based on the total number of mapped reads per replicate (reads per million
Translation values were calculated as a ratio of reads in the pulldown relative to reads in the input
Translation values were only included in the downstream analyses if the input contained greater than or equal to 100 unique reads across all replicates
Read counts across all fractions sequenced were normalized to account for differences in the number of reads sequenced across the different fractions
Normalized reads were derived by dividing each read count by the total number of mapped reads in that fraction
To get a normalized measure of distribution of reads across the polysome fractions
normalized read counts for each reporter was divided by the total normalized reads obtained for that reporter across all the fractions collected
the mean ribosome load (MRL) for a reporter was calculated as the sum of the product of polysome profile normalized read counts with the corresponding ribosome count
To determine whether there were systemic differences between NaP-TRAP-derived translation values and MRL
a linear regression was performed (y = NaP-TRAP translation
To examine the bias of different subsets of reporter mRNAs
the residual was calculated (residual = NaP-TRAP derived translation – predicted translation)
Difference in the culminative distributions of translation residuals were evaluated using a Mann-Whitney U-test for the following reporter mRNAs; reporters containing: (1) oORFs
Random forest regression models were employed to predict translation (scikit-learn 1.3; RandomForestRegressor)75
features were generated by one-hot encoding positions -6 to -1 and position +4 of each reporter sequence
k-mer counts (1-6 nucleotides) and uORF features were generated using a custom python script
features were filtered by calculating the Spearman Rank Correlation Coefficient (SPR) between each feature and translation prior to model training
Features that had a correlation greater than 0.05 or less than -0.05 were included in the random forest model
a permuted feature importance analysis was performed to identify the features with the greatest predictive power
structure) of reporter mRNAs was determined using the Vienna RNAFold program.57 Given that in silico RNA structure predictions are more accurate for shorter RNAs and the fact that the coding sequence and 3’ UTR of reporter mRNAs are identical
the first 200 nucleotides of each reporter were utilized to determine MFE
To investigate the effect of structure on translation the Pearson Correlation Coefficient was measured between translation (2 hpf and 6 hpf) and MFE
Reporters were divided further into groups based on whether they contained: no upstream start codons
For each group the correlation between structure and translation was determined
Reporters were ranked based on their translation at 2 and 6 hpf
Using the sum and difference of these rankings across timepoints
four groups of reporters were generated: (1) repressed
(3) repressed post-ZGA (active in HEK293T cells)
and (4) active post-ZGA (active in zebrafish)
Repressed and active reporters constituted the top and bottom 10% of reporters based on the sum of their ranks at 2 and 6 hpf
whereas the repress post-ZGA and active post-ZGA
were the top and bottom 10% of groups based on the difference between their ranks at 2 and 6 hpf
A differential motif enrichment analysis was performed on each group
Fold enrichment values were determined by dividing the count of each k-mer in the reporter group by the count of the k-mer in the library
whereas the significance of the fold-change was determined using a hypergeometric test (Bonferroni corrected p-value threshold)
To determine the regulatory potential of these motifs
reporters in the zebrafish 5’ UTR library enriched and depleted in each motif were identified (top and bottom 20%)
Enrichment and depletion of motifs were based on the sum of counts of kmers that were clustered to form each motif
A Mann-Whitney U-test was utilized to determine whether there was a significant difference in the distributions in the translation at 2 hpf
as well as the delta translation (translation 6 hpf / translation 2 hpf
and translation 6 hpf / HEK293T) for reporters enriched and depleted in each motif
To identify the top 10 motifs enriched for each of the RBPs tested
the rank of hexamers across each of the replicates were summed
The top 10 hexamers with the lowest summed rank
which were not enriched in the control groups of eCLIP experiments
A hypergeometric test was performed to determine whether the overlap between hexamers enriched in reporter groups described above and each of the RBPs was significant (Bonferroni corrected p-value threshold)
Candidate RBPs were assigned to motifs if they had an E-value was less than 0.05
For miR-430 the miRNA species with the highest complementary was selected for downstream analysis
Reporters with multiple seed sequences were excluded from the downstream analysis
The relationship between complementarity and change in translation (Translation 6 hpf / 2 hpf) was determined by calculating the Pearson’s Correlation Coefficient
To assess the importance of the microRNA seed
The complementarity analysis described above was repeated for reporters containing a seed mismatch for miR-430 (GCBCUU) and miR-1 (CAVUCC)
miR-1-1 / mir-1-2: 5’-UGGAAUGUAAAGAAGUAUGUAU-3’
To generate the feature rank plot (Fig. 6a)
features (k-mer counts of four nucleotides or fewer) were ranked based on the mean difference in translation between reporters enriched and depleted in the feature
Given the prominent effect of upstream open reading frames on translation
reporters with uORFs were excluded from the analysis
Features were only included in the analysis if there was a significant mean difference in translation at either timepoint (Mann-Whitney U test with Bonferroni corrected p-value threshold)
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article
All scripts used to process and analyze NaP-TRAP data will be released at the time of publication (https://github.com/ecstrayer/nap-trap_paper)
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Genome wide analysis of 3’ UTR sequence elements and proteins regulating mRNA stability during maternal-to-zygotic transition in zebrafish
A massively parallel 3’ UTR reporter assay reveals relationships between nucleotide content
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De Kumar from the Yale Center for Genome Analysis for sequencing support; S
Carmi for feedback on the manuscript; and all members of the Giraldez lab for feedback and support
We would like to acknowledge our funding sources: National Institutes of Health grant R01 HD100035 (A.J.G.)
National Institutes of Health grant R35 GM122580 (A.J.G.)
National Institutes of Health grant R00 HD093873 (J.-D.B.)
and National Institutes of Health grant R35 GM146883 (J.-D.B)
Department of Genetics and Genome Sciences
developed the pipeline to process and analyze NaP-TRAP data
on computational methods and supplied reading trimming and demultiplexing software
contributed experimental analysis and intellectual input
performed the sucrose gradient fractionation
and A.J.G are inventors on a provisional patent application filed by Yale University with the US patent office covering the NaP-TRAP method and the sequences described here
is founder of and has equity an equity interest in RESA Therapeutics
The remaining authors declare no competing interests
Nature Communications thanks Maria Barna and the other
reviewer(s) for their contribution to the peer review of this work
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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British author Nicolás Obregón has written three novels featuring half-Japanese inspector Kosuke Iwata: Blue Light Yokohama
and Black Suit City (set for release in 2019)
Andreas Neuenkirchen is the author of four novels
featuring Tokyo police inspector Yuka Sato
The two authors sat down to chat about their experience of writing crime fiction set in Japan
Andreas: In 2010 I approached publishers in Germany with a proposal for a series of crime novels set in Japan
and I was often told: “Japan is not a country people fantasize about” – which was news to me
More than one publisher seriously suggested changing the setting to Italy or Spain
and the nationalities of the protagonists accordingly
Did you have a similar experience when you pitched your first novel
Nicolás: I actually experienced quite the opposite
the first offer I received came from Germany
there still isn’t too much crime fiction set in Japan written by non-Japanese authors
I think maybe Blue Light Yokohama was something different
and it was written with English-language readers in mind
Andreas: I started writing the first novel in my series just before 3-11
Eventually I decided to set the plot a couple of weeks after the events
when they were still very much haunting people’s minds
and there were still minor power outages and supply shortages in Tokyo
Does it already feel like a purely historical event
Or did you consider it might be “too soon”
Nicolás: If I were going to write the book again today
Since 2011 was integral to the plot and timelines working out
I ran into the same problem: do I mention it or not
there might be a feeling that the story was happening in an alternative universe
I decided to include it without dwelling on the suffering and real events of that day
merely focusing on its impact on my own narrative
Andreas: Writing outside of the culture you were born into can be considered controversial these days
I’m waiting for the day someone comes up to me and says: “How dare you write about a female Japanese police inspector
and certainly not a police inspector?” Have you ever been accused of cultural appropriation
Nicolás: I’ve had a handful of complaints regarding my portrayal of Japanese culture
but never really about cultural appropriation
“Japanese people would never swear like that!” Which
One or two others have said I’ve misunderstood Japanese culture itself
But these are always non-Japanese people saying these things
I don’t think what we do is cultural appropriation
Did Agatha Christie steal the Belgian experience in the form of Poirot
Did Shakespeare steal the adolescent Italian experience in Romeo & Juliet
I sometimes get: “No Japanese will ever behave that way!” I find this kind of generalization much more offensive to the Japanese than anything I could ever write
If they were “normal” cops just working ordinary cases
It’s an odd contradiction; the need for “authenticity” when creating a fiction
while at the same time trying to thrill and transfix
I felt I had to invent an idealized police force
or at least a handful of ideal officers inside a far from ideal system
The actual Tokyo police is better known for forcing confessions and getting lost in bureaucracy than digging deep and investigating thoroughly
Nicolás: Over the course of my research I came across an academic study called “Contours of Police Integrity” by Carl Klockars and Sanja Kutnjak Ivković which examines police corruption and integrity across cultures
One of the findings was that while initially it appeared that Japanese police were far less corrupt than police in other countries
if you scratched the surface it became apparent that even when interviewed anonymously
Japanese police officers would not admit to corruption they had witnessed or been part of – instead tending to “toe the company line”
Andreas: Blue Light Yokohama refers to a song originally released in the 1960s
I was in a hotel room in Hiroshima and was thinking about writing a fiction loosely based on the unsolved Miyazawa family murder case from late 2000
“Blue Light Yokohama” was playing on the TV
It was one of those typical late-night commercials for CD box sets
I wrote down the words: “Blue Light Yokohama novel?” At first it just seemed a good working title
later I realized it was the heart of the novel
Andreas: You seem to have a nostalgic fascination with Japanese pop culture…
Nicolás: I watched a lot of anime when I was a kid
the shows I watched were dubbed into Spanish
but they couldn’t fool us: we could tell these stories were taking place somewhere far away – some place called Japan
Andreas: I also watched a lot of anime as a kid
but back in the 70s in Germany we were not aware that they were originally from Japan
More formative for my own interest in Japan were films like Akira or Tetsuo
Nicolás: I was especially fond of Captain Tsubasa
an anime show about a football prodigy growing up near Mount Fuji
pretty much all the shows my friends and I watched were Japanese
it wasn’t even the characters or the stories that fascinated me
The way people would become embarrassed at the most trivial things
Andreas: Which writers have influenced you
Nicolás: In terms of classic Japanese writers
I’d particularly highlight Points and Lines
In terms of contemporary Japanese crime writers
Out is one of my favorite books of all time
hatred — all of it set against a backdrop of economic downturn and the desperation of what it is to be a woman in an empty suburban existence
my all-time favorite book in the crime genre has to be The Secret in Their Eyes by Eduardo Sacheri
Andreas: Kirino is also one of my favorites
My major influences come more from the US than Europe or Asia
my uncle bought me novels by writers like Michael Connelly or Martin Cruz Smith
Nicolás: Martin Cruz Smith is such an important figure
he’s a good example for writing expertly outside of your own culture
Andreas: What are your plans for the Iwata series
Nicolás: The third novel [Black Suit City] will take Iwata back to Tokyo
and between Blue Light Yokohama and Sins as Scarlet he had quit the Tokyo Metropolitan Police and now works as a private investigator in LA
I can’t reveal too many details about the third Iwata novel except to say that it will be set just a few days before the 2020 Olympic Games
and it will center on the murder of an English girl found in a love hotel in San’ya
More about the authors at obregonbooks.com and www.andreas-neuenkirchen.asia
Senior Vice President Sales Leisure (left)
are looking forward to new successes and new milestones
GERMANY – SAWIKO, the AL-KO Vehicle Technology Group’s specialist for towbars and carrier systems for bicycles
“When we founded SAWIKO in 1995 as a small three-man business
we had a vision of where it would lead – today we offer the AL-KO Group’s complete portfolio for installation on motorhomes and caravans,” explained Guido Kovermann
founder of SAWIKO and Senior Vice President Sales Leisure at AL-KO Vehicle Technology:
we now serve a broad customer group: end customers
we have been assembling chassis for Tiny Houses and overseeing our online store activities in a purpose-built hall
we not only offer our customers first-class products
but also a premium service – and our continuous growth speaks for itself,” Kovermann said
Harald Hiller, president and CEO AL-KO Vehicle Technology, said: “The acquisition of SAWIKO in 2011 was a key strategic step in strengthening our presence in the European aftermarket. Since then, we have established nine customer centers across Europe
which has considerably strengthened our proximity to customers and partners
I would like to thank Guido Kovermann for his visionary spirit
senior vice president Sales & Marketing
added: “In his new expanded role as Senior Vice President Sales Leisure
Guido Kovermann now has overall responsibility for operational management
long-term strategic decisions and economic performance
His extensive industry experience and market knowledge make him the perfect person for the job.”
Kovermann reported: “We would like to celebrate our anniversary in style at the Caravan Salon in Düsseldorf this summer
I am gradually withdrawing from the operational business at SAWIKO due to my new role in the Group of Companies.”
our Neuenkirchen-Vörden site is particularly close to my heart – letting it go makes me a little wistful,” Kovermann continued
but I am delighted that the baton is being passed to a motivated
who has already been managing the SAWIKO business together with Kovermann for 18 months
will now take on overall responsibility for the Neuenkirchen-Vörden site
including the customer centers in Elsdorf near Cologne and Kleinkötz in Bavarian Swabia
who has been working with the AL-KO VT Group for ten years
started as a master’s student at the Paderborn site
then became head of the assembly department and from 2019 he was production manager
Read more about SAWIKO’s history here.
© 2023 G&G Media Group LLC
part of the city of DeKalb's public transit system
heads east on Lincoln Highway in downtown DeKalb Monday
VIRGIL TOWNSHIP – The driver of a car that crashed into a DeKalb-Northern Illinois University public transit bus Sunday in rural Virgil Township was flown to Loyola Medical Center in Maywood in critical condition
The driver is a 25-year-old St. Charles woman and was the only occupant of a 2021 Hyundai Venue, according to a news release from the Kane County Sheriff’s Office.
The Hyundai struck an NIU Huskie Bus, which is part of the city DeKalb public transit system, which merged with the NIU bus line in 2019. It had just picked up 22 passengers in Elburn on its third of four runs between DeKalb on a regular route for that day
DeKalb Transit Director Michael Neuenkirchen said Monday
as the typical ridership on that route comprises of two-thirds to three-fourths students coming back after the weekend
The Elburn transit route usually brings passengers between downtown DeKalb and the Metra train station in Elburn
“It’s one of our higher ridership days,” Neuenkirchen said
“Four people were taken to the hospital from the bus
None of them are in the hospital any longer
I am not aware of any major injuries that occurred
It was more for observation and to check out some of the pains reported at the scene.”
[ DeKalb public transit system to see route changes, including more shuttles to Elburn Metra station ]
The crash occurred on Route 38 and Francis Road at 8:39 p.m. Sunday as the bus was traveling west on Route 38, according to the release from the sheriff’s office.
The Hyundai was going north on Francis Road, came to a complete stop and then made a wide right turn onto Route 38, into the oncoming westbound lane in front of the bus, according to the release.
The bus was unable to avoid a collision, leaving the Hyundai with severe front-end damage, according to the release.
The driver of the Hyunda suffered multiple traumatic injuries and was initially taken by paramedics to Northwestern Medicine Delnor Hospital in Geneva. The woman was then flown to Loyola, according to the sheriff’s office.
The 2017 Gillig bus had front-end damage, which the transit agency mechanics are studying, Neuenkirchen said.
“At first glance, we don’t know if it’s fixable,” Neuenkirchen said. “We have very few accidents. Our drivers are always safety-minded. We’ve had hundreds of thousands of miles put on since 2019, so when this type of thing happens, it’s a big thing. Definitely not the norm.”
Thanks for visiting
ExpandMetra Trainman/Swingman Gordon Bowe works Metra's Union Pacific West line from Elburn to Ogilvie Transportation Center in Chicago in this Shaw Local file photo
A Chicago-based consultant is proposing a DeKalb city transit study in 2023 which would delve into whether a Metra route could connect directly to DeKalb
expanding residents’ access to the suburbs and Chicago
DeKALB – A Chicago-based consultant is proposing a DeKalb city transit study which would delve into whether a Metra route could connect directly to DeKalb
The DeKalb City Council is expected to vote on whether to hire Chicago-based consulting company Sam Schwartz Consulting for $98,739 to conduct a transit study to better determine how to expand and evolve the city’s existing public transit options
The vote is expected to take place during the Council’s regular meeting set for 6 p.m
DeKalb and much of the county lies about an hour or less west of downtown Chicago
and takes riders directly into the suburbs and downtown Chicago
DeKalb was once home to a train line to Chicago: The Chicago and North Western Transportation Company once operated commuter trains across the MidWest
“While people welcomed the tollway connection at the time, we now know the many downsides of an auto-centric world,” The Sam Schwartz Consulting company stated in the proposal
it has left DeKalb and NIU with only one way to connect students
[...] The Chicago metropolitan area is an undeniable economic powerhouse that DeKalb and NIU must leverage to remain competitive with the wider world.”
According to the transit study proposal submitted to the city by Sam Schwartz Consulting Dec
the study would help better aid the city’s plan to encourage growth both in the city and for Northern Illinois University enrollment
NIU has long struggled with enrollment over the past decade. Freshmen enrollment, however, has climbed over the past few years. This past fall, NIU defied national trends by recording an 11% increase in overall freshmen enrollment, according to first semester 2022-23 enrollment numbers.
city leaders including City Manager Bill Nicklas and Transit Director Mike Neuenkirchen recommend the City Council approve the contract
which would be paid for using the city’s Transportation Fund
which collects revenue from motor fuel tax
Elburn Metra Train Station Shaw Local file photo (Sandy Bressner) – A Chicago-based consultant is proposing a DeKalb city transit study in 2023 which would delve into whether a Metra route could connect directly to DeKalb
The city of DeKalb has already taken steps to better determine how area residents are using public transportation
City leaders also said they want to better determine residents’ desire for commuting by making the public bus to the Elburn Metra train station more frequent
the bus shuttle increased its routes from two trips in the morning and afternoon to five trips Monday through Friday
“The benefits of this connection include the diversification and growth of economic opportunity for DeKalb, greater access and resources for residents to reach employment and recreation opportunities in Chicago, and increased access for Northern Illinois University (NIU) to grow its talent, build institutional relationships, and empower commuting students, faculty and staff,” the proposal reads
The consulting company said that the study would also include studies to determine how potential railroad ridership fares and taxes would fuel DeKalb County and city revenue streams
Ridership fares for in-city public transit buses was reinstated in spring 2022, after a two-year moratorium on the fare because of the COVID-19 pandemic.
Correction: A previous version of this story misidentified the interstate that runs through DeKalb. It is Interstate 88. The story also incorrectly identified the direction that DeKalb is from Chicago and Elburn. DeKalb is west of both municipalities.
Today's print edition
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While vacationing in Saitama Prefecture recently
two cheerful-looking elderly gentlemen politely inquired about my nationality after complimenting my 5-year-old daughter's cuteness
Copenhagen!" The other one straightened his back
I smiled awkwardly and ushered my daughter away
hoping she hadn't picked up a new catchphrase (thankfully
she seemed entirely preoccupied with a group of mushrooms she had just detected on a nearby tree).googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); });
I realized that I was still silently fuming over the fact that I hadn't been a worthier warrior in the face of cultural stereotyping
All I could do is replay the scene with alternate endings in my head and vow to do better next time
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How an originally controversial form of Japanese nightlife entertainment became a worldwide phenomenon
although Western visitors are often surprised to find it practiced differently in its country of origin
The Japanese are not inclined to embarrass themselves drunkenly before a considerable audience of equally drunk strangers
they find privacy in so-called karaoke boxes
where one can face the music alone or among a select number of friends and family members
It is hard to pin down the one definite point in history when karaoke
Technical systems that might be considered predecessors of modern karaoke hardware were developed independently in at least five different parts of Japan during the 1960s
which is a composite of ‘kara’ (empty) and a shortened version of ‘orchestra,’ had then already been in use for some time
it did not refer to a form of entertainment for the masses but to professional singers using instrumental playback tracks for their performances when bands or orchestras could not be arranged
Karaoke was strictly a Japanese industry term before it became known as a global phenomenon
he started every morning by singing along to a radio program playing instrumental versions of pop songs
he was still singing when he entered his shop
Negishi requested him to connect a microphone to a tape deck so he could get a better idea of his vocal stylings
Unlike most laypeople, he did not recoil in horror from hearing his own voice. Negishi thought it was fun. And he was sure others would find it fun, too. He asked his staff to build a portable case for the machine and add a coin slot — there might be money in this contraption. Yes, it was the birth of… the Sparko Box
That was what Negishi called his invention
The Sparko Box was a cube measuring about 50 centimeters on each side
Its name was derived from a glass panel containing colorful lights adorning its front
Negishi asked an acquaintance working at NHK how to expand his repertoire of songs
The man told him he needed to find ‘karaoke tapes,’ using the industry term
He liked the word and intended to rename his machine accordingly
Eventually, Negishi distributed his Sparko Boxes to local bars. Customers were crazy about them. Traveling musicians weren’t. Bar owners often returned the devices after being harassed by entertainers seeing their livelihoods threatened. However, the box became a lasting hit in love hotels, primarily due to the colorful flashing lights on the front
which went well with the interior design commonly found in those places
Daisuke Inoue and Negishi have several things in common: They both invented early versions of karaoke machines
didn’t bother with having their inventions patented and didn’t call their creations karaoke
which first came to use in the snack bars and hostess clubs of Kobe’s Sannomiya entertainment district in the early 1970s
followed the basic principles of the Sparko Box
acknowledged and addressed the difficulties most non-singers have with singing popular songs
he recorded instrumental tracks in keys easier to master for casual revelers
While the 8 Juke faced the same opposition from disgruntled live performers as the Sparko Box
it eventually caught on and became a hit well past the Kobe city limits (Inoue hiring pretty hostesses to explain the concept helped)
Big consumer electronics companies like JVC jumped on the bandwagon and mass production started
yet another entrepreneur — Roberto del Rosario — invented what he called the Sing Along System (SAS)
it wasn’t that different from other systems already making the rounds
set Rosario apart from Inoue and Negishi: The Filipino businessperson did all the pesky paperwork and became the sole patent holder for everything karaoke
He reaped the rewards until his death in 2003
One thing was still missing when Japan entered its hard-partying bubble economy decade: subtitles
karaoke singers had to make sure they had the lyrics to their favorite tunes memorized or written down
far more short-lived invention that brought the words conveniently onto the screen: the LaserDisc
While the LP-sized predecessor of the DVD never caught on outside of Japan and a few other Asian markets
it was a LaserDisc-based karaoke machine from Pioneer that made this pastime even more accessible
it was karaoke that held that medium alive longer than one might think
The last documented LD title was produced in 2003
it needed to adapt to the tastes of younger generations
there was only one genre of music to sing — enka
folksy ballads preferred by older generations
savvy karaoke music licensers identified a star fit to lure in the kids
homegrown city-pop starlet but a man most often seen emphasizing his working-class roots in plain T-shirts and blue jeans — and he was born in the U.S.A
Japan was as crazy about Bruce Springsteen as the rest of the free world
and “Born in the U.S.A.” became the first contemporary pop-rock album to be fully licensed for karaoke
While karaoke spread into bars and nightclubs around the world
Japanese karaoke became a more private affair
more-or-less sound-isolated rooms became the norm
it’s not unusual for solitary patrons to sing entirely on their own
Professional performers like to take advantage of daytime discounts to hone their craft — belting it out in a karaoke box is less disruptive than doing it in your thin-walled rental apartment and less expensive than a proper rehearsal space
Disco lights illuminate modern karaoke rooms
drinks and snacks can be ordered by house phone (you might have to shout)
and rhythm instruments like tambourines and maracas are provided for extra fun
Karaoke equipment for home-use is widely available and the concept has spread to video games and mobile devices
While Negishi’s Sparko Boxes enjoyed considerable success
he decided the business was too much of a hassle and happily returned to assembling electronic devices for others
Inoue also turned his back on the hardware side of karaoke
at one point making a killing in music licensing
He also invented a detergent specifically for cleaning karaoke equipment
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Asahi Haikuist Network
Skeleton domein the burning sunA-bomb day--Satoru Kanematsu (Nagoya)
ocean foam--the memories that refuseto leave--Mona Bedi (Delhi
war newson a child’s ribswe read it--Justice Joseph Prah (Accra
garden rowsa strawberry seedunder the gum line--Anne Morrigan (Kitchener
back from the warroadside rose petalsshower upon him--Eugeniusz Zacharski (Darlowo
city riotersa wayside dandelionbends its head--Arvinder Kaur (Chandigarh
hearing cries for help--O violinist renew my faithno other theme but joy--Luciana Moretto (Treviso
there’s no going backonce you open to the worldpeony blossom--Noga Shemer (Storrs
heavenly river…the psychic’s nebulous wordsabout my future--Jackie Chou (Pico Rivera
------------------------------FROM THE NOTEBOOK------------------------------
stormy sealying for a momentbeside a dead jellyfish--Marcellin Dallaire-Beaumont (Brussels
Alexander Groth was pounded by the weight of rising ocean temperatures
El Nino--in my chest the soundof heavy rain
Sandra St-Laurent sprawled out in a sleeping position that resembled a star-shaped echinoderm
El Nino moonstranded on our bedsdreams of starfishes
Giuliana Ravaglia sketched parallel transverse lines in Bologna
the haikuist momentarily lost sight of a ship
rough sea--the flight of the dragonflyrising above the waves
rough seathe vanished steamerappears again
Padraig O’Morain reflected on the accuracy of circadian rhythms in Dublin
a cuckoo starts upthat’s all very wellbut you’re late today
Reflecting on Buddhist contradictions and the cycle of death and rebirth in Thames Ditton
Keith Evetts recalled asking a former ambassador to Tokyo about the burdens of representational entertainment
to which he replied: “There are times when a boiled egg in bed is absolute Heaven.”
sunny morningon the way to nirvanaa soft-boiled egg
Liz Gibbs had hoped for at least a tidbit of talk in Calgary
Maya Daneva briskly moved onto a new topic of discussion in The Netherlands
Isolationcraving the morselsof small conversation
seaside windjellyfish take overour conversation
Laurence Raphael Brothers reported that he “visited the charming and friendly but sadly diminished town of Soma in Fukushima and wandered into a tiny izakaya bar
where the barman regaled us with the story of his exploits in the annual capture-the-flag competition that commemorates the medieval rivalry between nearby towns.”
cavalry tacticsyield to izakaya cheer--fried food and sake
The haikuist ai li misses the arcades and food stands that lined a platform on pillars which stretched gaily from the shore into the sea
Tomislav Maretic walked to the cliff’s edge in Zagreb
Helga Stania found paradise stretched along a valley in the Swiss Alps
end of the path--the crickets’ song rising towardsthe Milky Way
narrow alleys’tween roof edges the starsabove the Engadine
Susan Rogers enjoyed a good show overhead Big Bear Lake
She remarked how the skies were so clear that “the heavens were indeed a river of jewels
summer fireworkshigh above thundering waves an explosion of stars
Sunflowers--may peace be restoredto Ukraine
stormy weatherno end in sightworld on shoulders
rough seas--waiting for her lovera girl at the quay
When he first saw a ship on the horizon off Hawaii
Eric Kimura said he “struggled a bit to capture it… then a week later” he saw a similar scene and realized that he “was missing the sense of touching the infinite
Balancing sereneYet on infinity’s edgeShip sails horizon
sparkling sea--sun behind the windowin the outline of infinity
Ed Bremson recalled a very enjoyable trip he took to the beach near Raleigh
a very long time ago--70 years to be exact
gazing over the sea,eating Frosted Flakesfrom the box
Julia Guzman kept a winter-themed travel journal while driving for days to Comodoro Rivadavia in Patagonia
sheep by the wire fencemorning Patagonian stormmidway on the trip
different cloud shapes--at the end of the long roadherd of guanacos
Keith Evetts sailed in a southerly direction
night passagethe Southern Crossbobbing up and down
Yutaka Kitajima can almost see the shoreline on an island in the Sea of Japan
Bedi recalled the taste of the Indian Ocean
morning seawe taste the salton our cookies
St-Laurent christened her busy roadside shop
fresh coat of painton the lemonade stand“El Nino”
Murasaki Sagano tapped her index finger approvingly at a fruit stand in Tokyo
Sweet soundsof the farmer’s fillipsfresh watermelons
Big pumpkinshared with a neighborhalf and half
Slobodan Pupovac can no longer pull in anything from Zagreb
Marek Printer can no longer hear sounds from an old pond in Kielce
looming El Ninothe frog burrows in muddeeper and deeper
seem to have grown tired of North Korea’s constant barrage of military projectiles
DeGuire may have knocked back a few shots to cool down
He suppressed his true feelings about the heat by using an expression from the military phonetic alphabet in Los Angeles
The rough seamissiles flying overSado island
ballistic rockets...and in the deep skya silent Milky Way
Sherry Reniker succumbed to the heat in Kent
in the Pacific againa “little boy”threatens
Refika Dedic stayed put in Bosnia and Herzegovina
tired of running awayI found refugein a lonely house
Wearing a light dress and sipping cold water
Angela Giordano nonetheless felt her body burn in Avigliano
Italy: from the window the African heat takes my breath away
crack in the chrysalis…letting bits of thisbody go
they spend their final days visiting flowers looking for pollen and nectar
Here’s a haiku about a colorful insect that C.X
Sunset glowin the distant westwarfare fires
Kobayashi Issa (1763-1828) may have faced the reality of death when he wrote this hokku: tooyama ga medama ni utsuru tombo kana
the distant mountains--reflected in the eyesof a dragonfly
----------------------------------------------------------------------------
Learn to let go at http://www.asahi.com/ajw/special/haiku/
The next issue of the Asahi Haikuist Network appears Aug
Readers are invited to send haiku related to this hokku by Kobayashi Issa: the distant mountains--reflected in the eyes of a dragonfly
on a postcard to David McMurray at the International University of Kagoshima
David McMurray has been writing the Asahi Haikuist Network column since April 1995
He is on the editorial board of the Red Moon Anthology of English-Language Haiku
columnist for the Haiku International Association
a column in The Language Teacher of the Japan Association for Language Teaching (JALT)
McMurray is professor of intercultural studies at The International University of Kagoshima where he lectures on international haiku
At the Graduate School he supervises students who research haiku
He is a correspondent school teacher of Haiku in English for the Asahi Culture Center in Tokyo
McMurray judges haiku contests organized by The International University of Kagoshima
McMurray’s award-winning books include: “Teaching and Learning Haiku in English” (2022); “Only One Tree Haiku
Music & Metaphor” (2015); “Canada Project Collected Essays & Poems” Vols
1-8 (2013); and “Haiku in English as a Japanese Language” (2003)
Information on the latest cherry blossom conditions
Please right click to use your browser’s translation function.)
A series based on diplomatic documents declassified by Japan’s Foreign Ministry
Here is a collection of first-hand accounts by “hibakusha” atomic bomb survivors
chefs and others involved in the field of food introduce their special recipes intertwined with their paths in life
A series about Japanese-Americans and their memories of World War II
In-house News and Messages
No reproduction or republication without written permission
I got into marathons as part of a midlife crisis
Now that I've transitioned comfortably into the routines of middle-age as I approach 50
I will fantasize a way to be a marathon runner without having to actually run a marathon
Half-marathons don't count; once you've done a full marathon its hard to find the glamour in something with "half" attached to the front
I need to go all the way.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); });
A Sungold filly and Floriscount colt became the winners of the Foal Selection Show at Hof Kasselmann in Hagen
Eight fillies and ten colts qualified for the 2011 German Foal Championships held in Lienen
The champion filly was a Sungold x Davignon offspring
This talented young mare scored 81,5 points and left a Fidertanz x Sandro Hit filly behind her
She is bred by Frank Moormann and scored 79,5 points
The champion colt was a chestnut Floriscount x Sunny Boy offspring bred by Ludger Emke from Cloppenburg
His colt scored 84 points and referred Johannes Westendarp's Fürstenball x Wolkentanz II colt to a second place with 81,5 points
Kerstin Klieber acquired the champion colt on the spot
Another Furstenball offspring placed third: He is bred by Alfons Brünen out of a Sandro Hit dam
Photo © Kiki Beelitz
Fillies - Qualified for the 2011 German Foal Championship
Colts - Qualified for the 2011 German Foal Championship
Related LinksRomanov and Diamond Hit Produce Winners at 2011 Lodbergen Foal Selection ShowEurodressage Stallion at Stud: Furstenball
Stalls for Rent at Durondeau Dressage in Peer, Belgium
Exceptionally Well Located Equestrian Facility in Wellington, Florida
Well-built Equestrian Estate With Multiple Business Opportunities in Sweden
Stable Units for Rent at Lotje Schoots' Equestrian Center in Houten (NED)
For Rent: Several Apartments and Stable Wing at High-End Equestrian Facility
Stable Wing Available at Reiterhof Wensing on Dutch/German border
Real Estate: Well-Appointed Country House with Extensive Equestrian Facility in the U.K.
Rémi Blot
With almost 56,000 convenience stores in Japan today
it’s hard to believe everything started with a single shop
With their familiar logos shining their lights on every street corner and many places in between
it’s also hard to believe that this first effort didn’t even have a big franchise behind it
And with convenience stores shifting over ¥10 trillion per year
it seems unimaginable that the concept wasn’t a rousing success from the get-go
It didn’t carry any of those logos that guide us through the night today
it didn’t sell spam-filled onigiri or microwaveable yakisoba sandwiches and you couldn’t pay your health insurance or your Uniqlo online order at the counter — all things a modern convenience store customer now takes for granted
Editorial credit: Wiennat M / Shutterstock.com
The history of what we nowadays consider a Japanese convenience store
Japanese department store chain Ito-Yokado bought franchise rights for the American brands of 7-Eleven convenience stores and Denny’s family restaurants (another contemporary Japanese mainstay far removed from its US roots)
Building smaller shops in residential neighborhoods helped the company branch out into areas where laws kept it from building bigger outlets
what was convenient to Americans wasn’t too attractive to the Japanese
primarily by taking over existing businesses
That began to change when the onigiri entered the scene
Where there were usually hot dogs at the register
The traditional rice snack dating back to the Heian Era (794 to 1185) was easy to prepare and could be sold cheaply
It conveyed a sense of homey familiarity to local customers
yet it offered the potential for constant innovation with its endless variations of fillings
the average 7-Eleven store sells about 200 these days
Which flavor dominates those sales is a well-kept secret
but the famous tuna-mayonnaise filling introduced in 1983 might give the more conservative salmon a run for its money
the child of an employee came up with the recipe
Editorial credit: Dutchmen Photography / Shutterstock.com
The onigiri’s success hinged not only on its tastiness but also on fast delivery
7-Eleven Japan radically upgraded its inventory system over time
reducing the turnaround between store order and delivery from one week to 24 hours
This paved the way for further Japanification of the konbini product
Selling the winter staple of oden as well as yakitori and other ready-to-eat chicken products at the counter became standard
The innovative Japanese convenience stores were thriving
The franchisee bought the franchiser by acquiring a majority in shares
What seemed an unshakable all-American brand effectively became a Japanese company
It went into an international acquisition frenzy lasting several years
resulting in the organization’s rebranding as “7 and i Holdings” in 2005
it also owns the Sogo and Seibu department stores
Editorial credit: Karolis Kavolelis / Shutterstock.com
Almost 50 years after its first appearance on Japanese soil
7-Eleven is far from the only konbini game in town
If you want to survive as a convenience store franchise
you better find a unique selling point to set your brand apart
FamilyMart itself was bought by garish knick-knack store chain Don Quijote in 2020 but maintained its branding)
While 7-Eleven is known for its tuna-mayo onigiri and FamilyMart wouldn’t be FamilyMart without fried Fami-Chicken
Lawson carves out a niche with leaner and somewhat healthier food options under their Natural Lawson banner
Selling products that can be a bit pricier and hopefully of higher quality has become Lawson’s strategy
The brand suffered an infamous incident in 2002
giving a whole new meaning to the term ‘finger food,’ when a woman in Sendai found a severed fingertip in a Lawson rice ball
originally belonging to an onigiri plant worker (the fingertip
Editorial credit: Ned Snowman / Shutterstock.com
originated in the US —and also became an entirely Japanese-owned operation
So much so that there are no longer any Lawson stores left in the US (a Hawaiian mini-revival in 2012 was short-lived)
while they are still going strong in Japan
with its western-movie font and depiction of a vintage milk can
Lawson was a milk farmer from Ohio who opened his first shop in 1939 to sell his dairy products directly to customers
7-Eleven is the most successful of the konbini chains in Japan
According to an extensive survey from the NPO Japan Productivity Center (JPC)
In Hokkaido, Seicomart is king — both in terms of business success and customer satisfaction
Most items are manufactured under their own label Secoma
using local ingredients to create products you won’t find anywhere else
Saitama is the only place outside of Japan’s northernmost prefecture to have a couple of Seicomart stores
Seicomart is also known for having a big heart for foreigners (and their money)
offering currency exchange and the kind of imported junk food that every non-Japanese craves sooner or later
Editorial credit: Terence Toh Chin Eng / Shutterstock.com
the convenience store boom has slowed down a little bit
Market saturation and competition from online shopping and food delivery services are taking their toll
certain age-old practices are being questioned
does every shop everywhere have to be open 24 hours a day
The Japanese affectionately call them reizoko no kawari (meaning substitute fridges)
Although that’s hardly the only thing they substitute
you can even get documents concerning your tax payments and residency from machines at convenience stores
No need to pick a number and grab a seat at your municipal office anymore
And while you are typing in your passcodes
you might as well munch on a tuna-mayo onigiri
Returningsnow white ibisesto Sado--Satoru Kanematsu (Nagoya)
one by one,the turkey buzzards return...lingering snow--Joshua Gage (Cleveland
snow-free Decemberforest feeds the deerI feed my dark mood--Nuri Rosegg (Oslo
winter flu--the endless white ofmy notebook--Alexander Groth (Neuenkirchen
breathing it all incold-brewed wintermorn--Roberta Beach Jacobson (Indianola
broken branchesmaking him a hot chocolatefor a start--Marie Derley (Ath
Mueller’s electric tea kettle:skipping two stages of boilingright to the ‘wild billows’--Patrick Sweeney (Pittsburgh
deep snow--the daruma doll’sunpainted eye--Farah Ali (Brighton
Illusionist’s eyesI sure will not follow themhe sure knows I will--Horst Ludwig (Seattle
alone by the springthat stirred me and this riverI write my last line--James Penha (Bali
Tsanka Shishkova offered this line from Sofia
Bulgaria: deep darkness in my dream the day dawns
Groth embraced a child in the throes of a seizure
febrile convulsion--this cozy coldnessof your hugs
Charles Smith wrote this haiku while thinking about an exhibition of photographs entitled “Human Shadow Etched in Stone” at Hiroshima Peace Memorial Museum
Dennis Woolbright coached 14-year-old Bao Le in Vietnam
Soft breeze,dying banyan tree,stay singing sparrows
A drop of sleet fell on John Hawkhead in Bradford on Avon
sprinkling eartha chaffinch releasesits song of rain
Huffman calmed her nerves while driving down the road in Wilkesboro
wipers swish swipehypnotic erasureabusive spouse
Writing from snow-covered mountains in Joetsu
Yutaka Kitajima composed a haiku based on Kenji Miyazawa’s 1922 poem “Eiketsu no Asa” about his sister Toshiko murmuring “Get me some sleet
please” (ameyuju totechite kenja) before dying of tuberculosis at the age of 25
Her final faint smileover fresh sleet in the bowl...heavenly ice cream
Stania was chilled listening to Franz Schubert’s 1827 piano sonata in the cold and dark landscape of her winter home
Huffman’s garden concert leaving her deck scattered with dead katydids
A fiddlers’ green alludes to an after-life where there is perpetual mirth
a fiddle that never stops playing and dancers who never tire
cold snapgreen fiddlers frozensymphony silenced
David Cox recoiled at the touch of something misconceived
with his death--leaving the eartha white moth
Commenting on a work of improvisation in Minneapolis
Jerome Berglund riffed on the most interesting-looking moth in the world that was first described by the British entomologist Frederic Moore in 1882
Picasso mothgrand architecturemeans bad policy
Mirela Brailean grieves the discordance between now and then in Iasi
the end of the yearon mom’s wall calendarit’s still summer
the postponement of what would have been a normal encounter for late summer until late November
“it was a weird moment--my shift at an end
finished for the dayI follow a mosquitodown the dark stairwell
John Pappas shrugged his shoulders and disappeared underground in Boston
T’Kelah Smith’s spoken verse turned white overnight in Tchula
stale coldnipping at my noseglimpse of breath
Kanematsu heard all kinds of sounds outside his kitchen window
Hearing aidamplified farewelllast crickets
Deep autumn--the next door neighborunemployed
Diana Silver penned this haiku while traveling across Japan
Kathabela Wilson restored herself during a Japanese cultural meeting at Storrier Stearns Japanese Garden in Pasadena
StratocumulusColourless morning in bedOutside gold leaf falls
this kintsugi lifeso many breaks nowmy heart turns gold
Kiyoshi Fukuzawa penned this haiku while convalescing in Tokyo and thinking about his youth spent in Arab countries
Dark clouds all overI miss the Arabian sandshospital window
thanksgiving tripmore dead deeralong interstate
Tanja Trcek hiked an autumn trail with a partner in Golnik
Mircea Moldovan spotted a white fur coat in Letca
Anne-Marie McHarg strolled through the campus of Queen Mary University of London
a white rabbiton the endless expansefirst snow
A meditative walkThrough shadowy treesAutumn moon
Murasaki Sagano was drawn to a secretive place away from prying eyes in Tokyo
Levko Dovgan felt melancholic while hunting for red leaves in Lviv
Shishkova brush-stroked this line at home: painting moonlit red maple leaves in a glass vase
When deciduous trees take on autumnal colors
the evergreen bamboos become freshly green as if it were spring
McHarg mixed and matched sprays of yellow and green
Cox pulled down the earflaps on a fur-lined cap
Green bamboo foliageAnd a winter jasmineIkebana
Evergreen bonsaiarranged on the porch--ready for winter
green barley shootsit’s the season fortourist hats
Autumn rosesobscure my sorrowin my own time
Junko Saeki wrote: “as I approach the end of my journey
memories of people who accepted me and gave me unconditional love and helped me believe in myself and others
high skies of autumnfaces of peopledearest to me
Arvinder Kaur never had a chance to say goodbye in Chandigarh
Berglund suggested he was nonetheless satisfied
John Hamley has lived a good life in Marmora
Sagano plans to tend her garden until the end
Luminita Suse brushed snowflakes off velvety red rose petals in Ottawa
Feel the windthis withered fieldhas seen it all
See to itthat a red rose still bloomsthe end of the year
Nazarena Rampini admired her little winter garden in Pogliano Milanese
Decemberwith thin blades of frostthe embroidered pine
Natalia Kuznetsova sat by her father’s bedside in Moscow
on his deathbeddad asks about his roses...falling leaves
If Mario Massimo Zontini were able to choose
he knows when he would like to lay his head down on a satin pillow and dream forever in Parma
when my time comesbe it early in the yearwhen the wintersweet blooms
“The Pillow Book” (Makura no Soshi) penned by Sei Shonagon in 1002 during the Heian Period (794-1185) is a list of musings about such as things as those who have lost their power: “A woman who has taken off her false locks to comb the short hair that remains.”
Hawkhead conjured this line of snow: pillow talk dreams of snow clouds
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The next issues of the Asahi Haikuist Network appear on Dec
Readers are invited to send haiku about those who have lost their power on a postcard to David McMurray at the International University of Kagoshima
tackles with targeted physical use and quick starts in the sand – on Saturday
the beach rugby tournament in the SportBeachArena will be action-packed
Eight women's and just as many men's teams compete against each other in the full-contact sport on the sand
The tournament is organized by Rugby Rostock – Dierkower Elche and the university sports department at the University of Rostock
teams with five players each compete on the 30 by 15 meter playing field
The rugby teams have to act quickly in a relatively short time
grab the rugby ball with its rotationally elliptical shape from the opposing team and stop them by tackling or holding on
“The biggest challenge in beach rugby is running on the sand
“It’s exhausting to the tenth degree,” says tournament organizer André Goeda
The great interest shown by rugby players in the event shows how popular the tournament is in Warnemünder Sand
The entry list for the beach rugby tournament during the 84th edition was already in March
Warnemünder Woche full and the event “sold out”
“The many early reports show how happy everyone is to finally be able to play sports together again and meet again at events,” says Goeda
explaining the good turnout for the tournament
Since the theme of the closing party is “Partyanimals,” he certainly expects that some teams may appear at the tournament in costume
the sporting idea would be in the foreground
The following teams compete in beach rugby on the Baltic Sea beach: the men's teams Siemensstadt
1860 Bremen and Rostock as well as the women's teams St
the Olympic idea is more important: being there is everything!” says Goeda
At the beach rugby tournament as part of the Warnemünder Woche In 2019
the Rostock teams (University of Rostock/Dierkower Elche) were each in second place
The men from Berlin won the men's event and the Dortmund women won the women's event
Mecklenburg-Vorpommern's largest sailing event and second largest folk festival
the aim is to offer a diverse programme on
life at sea and the ports of the world were yesterday
WARNEMÜNDER WOCHE officeRostock & Warnemünde Tourism OfficeAm Strom 5918119 Rostock-Warnemünde
Phone: + 49 (0) 381 / 548 00 23Email: warnemuenderwoche@rostock.de
Apparently there is a trailer out there for an upcoming superhero movie that features scenes in an Asian city that is supposed to be Tokyo
Before I even got a chance to see the clip
of course) of how this Tokyo doesn't look anything like the real Tokyo
it looks like Tokyo imagined by somebody who has never been there.googletag.cmd.push(function() { googletag.display('div-gpt-ad-1499653692894-0'); });
I still haven't seen the trailer in question — actually
I've already forgotten which film it is for — but I have a pretty good idea what this Tokyo looks like: thousands of colorful neon lights reflected on black asphalt wet with rain
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Partner Giles Salmond and associate barrister Guy Bud examine the recent decision in Thyssenkrupp Materials (UK) Ltd v Revenue and Customs Comrs [2024] UKUT 00079 (TCC)
in which the Upper Tribunal found against HMRC’s strict interpretation of the inward processing relief procedure
It is no secret that importers and customs agents are often required to provide vast amounts of complex and detailed information to HMRC
The process can be daunting and difficult to navigate
It is practically inevitable some human errors will creep in
The Upper Tribunal’s decision in Thyssenkrupp Materials (UK) Ltd v Revenue and Customs Comrs [2024] UKUT 00079 (TCC) will help those who deal with customs processes better understand what the acceptable margins of error are
The case is also a salutary lesson that you should not always take what HMRC throws at you at face value
there may be strong arguments to challenge HMRC’s decision
Where no special customs procedure or exemption applies
customs duty and import VAT become payable once goods are imported into the UK
inward processing (“IP”) enables an importer to suspend such liabilities for goods imported specifically for “processing” ahead of re-export or some form of processing to allow the importer to claim relief from import duties (known as “IPR”)
There are elaborate formalities for using IP designed to ensure goods do not enter free circulation without payment of duties and VAT
It is worth bearing in mind that this is the principal objective of the customs rules
and the relevant IP authorisation contains detailed requirements for the importer to follow to allow the customs authorities to supervise duty-suspended goods
correctly discharging these formalities can be vital for businesses that import goods and are able to use IP or other customs procedures
These rules are particularly pertinent to those in the manufacturing
but the procedures are used for a myriad of different businesses involved in importing goods
The facts in Thyssenkrupp were similar to those facing many importers
The taxpayer’s business imported aluminium and other materials into the UK in 2014 in order to produce aircraft components
It was authorised to use IP with a “throughput” period of six months and was required to submit a Bill of Discharge (“BoD”) each quarter
which would allow HMRC to verify that the relevant conditions had been met
The taxpayer in this case was required to submit quarterly BODs in Excel spreadsheet format
showing that goods imported under the IP authorisation had been discharged from the relief
Each BoD contained between 100,000 and 200,000 data points and related to a large number of importations
HMRC identified information in the BoD that was inconsistent with the information entered in HMRC’s Management Support System (“MSS”) and issued a Post-Clearance Demand Note demanding suspended taxes of nearly £9m
During the period in dispute (which preceded the UK’s departure from the European Union)
the conditions under which an import tax debt could arise under IP were drawn directly from EU law in the form of Article 254 of the Community Customs Code (“CCC”)
This provided that a customs debt would be incurred as a result of failure to comply with “obligations” or “conditions” unless any such failure had “no significant effect on the correct operation of the […] customs procedure in question”
There was further guidance in Regulation (EEC) 2454/93 (“the Implementing Regulation”)
The First-Tier Tribunal (“FTT”) sided with HMRC
drawing heavily from C-262/10 Döhler Neuenkirchen GmbH v Hauptzollamt Oldenburg (2012) ECLI:EU:C:2012:559 decided by the Courts of Justice of the European Union (“CJEU”)
or any mismatch between the BoD and the MSS
The FTT also held that a single error in an import or disposal line of one import entry on a BoD means that customs duty and import VAT are due on all the goods covered by the BoD.”
This was a strict interpretation of the legislation and case law and would have extremely far-reaching implications for all importers using IP and other special procedures
Considering the sheer number of data points on a complex BoD
a certain number of human errors are almost inevitable
The FTT decision implied that one alone could be sufficient to trigger a significant tax charge
Many importers will be relieved to know that the UT disagreed with the FTT and struck a much more moderate stance
Reviewing the CJEU’s historical case law on documentary errors in detail
it distinguished Döhler on the basis that it dealt with a case in which the taxpayer had failed to file any BoD at all within the prescribed time limits
or even after those time limits had been notified to the taxpayer and extended by the customs authorities
that any documentation-related failure on a BOD must have comparable effects to those found in Döhler
where no BOD was filed at all (despite having been warned of this requirement)
“There is no dispute that the IPR regime requires strict compliance with the obligations for a relevant trader’s authorisation
and that non-compliance with those obligations may lead to a customs debt being incurred in relation to the relevant imported goods
The proposition that a single error in a single cell within a BoD will lead to a customs debt being incurred in relation to all goods covered by that BoD is
so disproportionate as to be entirely absurd
and we do not consider that it is supported by the Döhler judgment.”
the UT pointed to a “fundamental difference” between failure to file a BoD and filing a BoD with a small number of inadvertent and non-fundamental errors
The UT said: “The latter does not come close to representing a document that is (to adopt by analogy the words of Advocate General Slynn in [C-121/87] Bayernwald Früchteverwertung) is ‘so incomplete and inaccurate’ that it cannot fairly be described as a BoD at all.” Assuming one had been filed that met this standard
it was clear an inaccuracy would not invalidate the entire BoD
The UT also rejected the FTT’s view that the BoD were required to contain accurate particulars allowing immediate reconciliation of the BoD data with the data held in HMRC’s MSS
This means that an error on the BoD would not trigger a charge to customs duty even in relation to the specific entry to which it relates
Although concerned with a legal framework that has changed due to Brexit
the UT decision in Thyssenkrupp remains relevant and will be a major relief for many importers and businesses that regularly rely upon IP and other special procedures
The UT’s extensive consideration of how different kinds of error should be interpreted will also be important
Import taxes remain an often-misunderstood area
and expert advice should be sought as soon as any possible dispute arises with HMRC
Although this case shows that certain errors do not lead to a customs debt
prudent to adhere to authorisations carefully
where HMRC does take issue with non-compliance with customs formalities
there may be scope to challenge assumptions made by HMRC successfully
You can find further information regarding our expertise, experience and team on our Tax Litigation and Resolution page
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He is best known for his work on high-temperature superconductivity
which earned him the Nobel Prize in Physics in 1987
He was born on 16 May 1950
He received his undergraduate degree in physics from the University of Münster in 1972 and his Ph.D
he worked as a researcher at the IBM Research Laboratory in Ruschlikon
discovered high-temperature superconductivity in a new class of materials called cuprates
This discovery was a major breakthrough in the field of superconductivity
as it allowed for superconductivity to occur at much higher temperatures than previously thought possible
Bednorz has continued to work on the development of new materials with novel electronic properties
He has also been involved in the design and fabrication of new devices based on these materials
such as superconducting sensors for medical imaging
Georg Bednorz’s work has had a profound impact on the field of materials science and has opened up new avenues for research and development in a wide range of fields
In 1987, Bednorz and Muller were jointly awarded the Nobel Prize in Physics for their discovery
This made Bednorz the youngest person ever to receive the Nobel Prize in Physics at the time
He has received numerous other honors and awards throughout his career
including the German Order of Merit and the Benjamin Franklin Medal in Physics
His discovery has had a profound impact on materials science and has opened up new possibilities for the development of technologies such as power transmission and medical imaging
Bednorz continues to work as a physicist and is currently a director at the Max Planck Institute for Solid State Research in Stuttgart
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