bluuebunny/crossref_metadata_2025 huggingface dataset:
Strategic Planning and Essential Steps for Establishing a Uterine Transplant and Rehabilitation Program
Authors: Dorafshar Amir H. , Hamidian Jahromi Alireza , Horen Sydney R. , Schechter Loren S. , Johannesson Liza , Testa Giuliano , Hertl Martin , Dewdney Summer , Aschkenasy Jeannie , Wood-Molo Mary , Brincat Cynthia , Cherullo Edward , Behel Jay M. , Hebert Charles , Shulman Robert , Bassi Shruti , Alecci Anna T. , Konety Badrinath
Published: November 2021

UTx is performed to address absolute uterine infertility in the presence of uterine agenesis, a nonfunctional uterus, or after a prior hysterectomy. After the initial success of UTx resulting in a livebirth (2014) in Sweden, there are over 70 reported UTx surgeries resulting in more than 40 livebirths worldwide. Currently, UTx has been performed in over 10 countries. As UTx is transitioning from an “experimental procedure” to a clinical option, an increasing number of centers may contemplate a UTx program. This article discusses essential steps for establishment of a successful UTx program. These principles may be implemented in cis- and transgender UTx candidates.
Circumferential Resection Margin is Associated With Distant Metastasis After Rectal Cancer Surgery
Authors: Agger Erik , Jörgren Fredrik , Lydrup Marie-Louise , Buchwald Pamela
Published: November 2021

Objective:

To evaluate circumferential resection margin (CRM) as a risk factor for distant metastasis (DM) in rectal cancer.

Summary of Background Data:

The treatment of rectal cancer has evolved over the last decades. Surgical radicality is considered the most important factor in preventing recurrences including local and distant. CRM ≤1.0 mm is considered to increase recurrence risk. This study explores the risk of DM in relation to exact CRM.

Methods:

All patients treated with abdominal resection surgery for rectal cancer between 2005 and 2013 in Sweden were eligible for inclusion in this retrospective study. Primary endpoint was DM.

Results:

Twelve thousand one hundred forty-six cases were identified. Eight thousand five hundred ninety-three cases were analyzed after exclusion. Seven hundred seventeen (8.6%) patients had CRM ≤1.0mm and 7577 (91.4%) patients had CRM >1.0 mm. DM recurrence rate at 5 years was 42.1% (95% CI 32.5–50.3), 31.5% (95% CI 27.3–35.5), 25.8% (95% Confidence Interval (CI) 16.2–34.4), and 19.5% (95% CI 18.5–19.5) when CRM was 0.0 mm, 0.1 to 1.0 mm, 1.1 to 1.9 mm, and CRM ≥2mm, respectively. Multivariable analysis revealed higher DM risk in CRM 0.0–1.0 mm versus >1.0 mm (hazard ratio 1.30, 95% CI 1.05–1.60; P = 0.015). No significant difference in DM risk in CRM 1.1–1.9 mm versus ≥2.0 mm (hazard ratio 0.66, 95% CI 0.34–1.28; P = 0.224) could be detected.

Conclusions:

The risk of DM decreases with increasing CRM. Moreover, CRM ≤1.0 mm is a significant risk factor for DM. Thus, CRM is a dominant factor when discussing risk of DM after rectal cancer surgery.
Performance of the 7th and 8th Editions of the American Joint Committee on Cancer Staging System in Patients with Intraductal Papillary Mucinous Neoplasm-Associated PDAC
Authors: Margonis Georgios Antonios , Pulvirenti Alessandra , Morales-Oyarvide Vicente , Buettner Stefan , Andreatos Nikolaos , Kamphues Carsten , Beyer Katharina , Wang Jane , Kreis Martin E. , Cameron John L. , Weiss Matthew J. , Soares Kevin , Fernández-del Castillo Carlos , Allen Peter J. , Wolfgang Christopher L.
Published: November 2021

Objective:

To validate the 7th and 8th editions of the AJCC staging system for patients with invasive carcinomas arising in association with IPMN (IPMN-associated PDAC).

Background Data:

Although several studies have validated AJCC systems in patients with conventional PDAC, their applicability to IPMN-associated PDAC has not been assessed.

Methods:

Two hundred seventy-five patients who underwent resection for IPMN-associated PDAC between 1996 and 2015 at 3 tertiary centers and had data on the size of the invasive component and lymph node status were identified. Concordance probability estimates (CPE) were calculated and recursive partitioning analysis was employed to identify optimal prognostic cutoffs for T and N.

Results:

The CPE for the 7th and 8th editions of the AJCC schema were relatively good (0.64 for both) and similar for colloid and tubular subtypes (0.64 for both). The 8th edition introduced T1a sub-staging and a new distinction between N1 and N2. The utility of the former was confirmed, although the latter did not improve prognostic discrimination. The successful validation of the 8th edition of the AJCC criteria in patients with tubular and colloid subtypes allowed us to compare these patients in early vs late T and N stages which showed that with advanced disease, the prognostic superiority of colloid tumors over their tubular counterparts diminishes.

Conclusions:

Our findings support the use of the AJCC 8th edition in the IPMN-associated PDAC population, but suggest that certain cutoffs may need to be revisited. In advanced AJCC stages, patients with colloid vs tubular subtypes have comparable prognosis.
Plasma and wound fluids from trauma patients suppress neutrophil extracellular respiratory burst
Authors: Kim Hyo In , Park Jinbong , Konecna Barbora , Huang Wei , Riça Ingred , Gallo David , Otterbein Leo E. , Itagaki Kiyoshi , Hauser Carl J.
Published: November 2021

BACKGROUND

Trauma increases susceptibility to secondary bacterial infections. The events suppressing antimicrobial immunity are unclear. Polymorphonuclear neutrophils (PMNs) migrate toward bacteria using chemotaxis, trap them in extracellular neutrophil extracellular traps, and kill them using respiratory burst (RB). We hypothesized that plasma and wound fluids from trauma patients alter PMN function.

METHODS

Volunteer PMNs were incubated in plasma or wound fluids from trauma patients (days 0 and 1, days 2 and 3), and their functions were compared with PMNs incubated in volunteer plasma. Chemotaxis was assessed in transwells. Luminometry assessed total and intracellular RB responses to receptor-dependent and independent stimulants. Neutrophil extracellular trap formation was assessed using elastase assays. The role of tissue necrosis in creating functionally suppressive systemic PMN environments was assessed using a novel pig model where PMNs were incubated in uninjured pig plasma or plasma from pigs undergoing intraperitoneal instillation of liver slurry.

RESULTS

Both plasma and wound fluids from trauma patients markedly suppress total PMN RB. Intracellular RB is unchanged, implicating suppression of extracellular RB. Wound fluids are more suppressive than plasma. Biofluids suppressed RB maximally early after injury and their effects decayed with time. Chemotaxis and neutrophil extracellular trap formation were suppressed by biofluids similarly. Lastly, plasma from pigs undergoing abdominal liver slurry instillation suppressed PMN RB, paralleling suppression by human trauma biofluids.

CONCLUSION

Trauma plasma and wound fluids suppress RB and other key PMNs antimicrobial functions. Circulating suppressive signals can be derived from injured or necrotic tissue at wound sites, suggesting a key mechanism by which tissue injuries can put the host at risk for infection.
Intercostal liposomal bupivacaine injection for rib fractures: A prospective randomized controlled trial
Authors: Wallen Taylor E. , Singer Kathleen E. , Makley Amy T. , Athota Krishna P. , Janowak Christopher F. , Hanseman Dennis , Salvator Ann , Droege Molly E. , Strilka Richard , Droege Christopher A. , Goodman Michael D.
Published: November 2021

BACKGROUND

Blunt chest wall injury accounts for 15% of trauma admissions. Previous studies have shown that the number of rib fractures predicts inpatient opioid requirements, raising concerns for pharmacologic consequences, including hypotension, delirium, and opioid dependence. We hypothesized that intercostal injection of liposomal bupivacaine would reduce analgesia needs and improve spirometry metrics in trauma patients with rib fractures.

METHODS

A prospective, double-blinded, randomized placebo-control study was conducted at a Level I trauma center as a Food and Drug Administration investigational new drug study. Enrollment criteria included patients 18 years or older admitted to the intensive care unit with blunt chest wall trauma who could not achieve greater than 50% goal inspiratory capacity. Patients were randomized to liposomal bupivacaine or saline injections in up to six intercostal spaces. Primary outcome was to examine pain scores and breakthrough pain medications for 96-hour duration. The secondary endpoint was to evaluate the effects of analgesia on pulmonary physiology.

RESULTS

One hundred patients were enrolled, 50 per cohort, with similar demographics (Injury Severity Score, 17.9 bupivacaine 17.6 control) and comorbidities. Enrolled patients had a mean age of 60.5 years, and 47% were female. Rib fracture number, distribution, and targets for injection were similar between groups. While both groups displayed a decrease in opioid use over time, there was no change in mean daily pain scores. The bupivacaine group achieved higher incentive spirometry volumes over Days 1 and 2 (1095 mL, 1063 mL bupivacaine vs. 900 mL, 866 mL control). Hospital and intensive care unit lengths of stay were similar and there were no differences in postinjection pneumonia, use of epidural catheters or adverse events bet ween groups.

CONCLUSION

While intercostal liposomal bupivacaine injection is a safe method for rib fracture-related analgesia, it was not effective in reducing pain scores, opioid requirements, or hospital length of stay. Bupivacaine injection transiently improved incentive spirometry volumes, but without a reduction in the development of pneumonia.

LEVEL OF EVIDENCE

Therapeutic/care management, Level II.
Comprehensive and accurate genetic variant identification from contaminated and low-coverage Mycobacterium tuberculosis whole genome sequencing data
Authors: Heupink Tim H. , Verboven Lennert , Warren Robin M. , Van Rie Annelies
Published: November 2021

Improved understanding of the genomic variants that allow

Mycobacterium tuberculosis

(Mtb) to acquire drug resistance, or tolerance, and increase its virulence are important factors in controlling the current tuberculosis epidemic. Current approaches to Mtb sequencing, however, cannot reveal Mtb’s full genomic diversity due to the strict requirements of low contamination levels, high Mtb sequence coverage and elimination of complex regions. We have developed the XBS (compleX Bacterial Samples) bioinformatics pipeline, which implements joint calling and machine-learning-based variant filtering tools to specifically improve variant detection in the important Mtb samples that do not meet these criteria, such as those from unbiased sputum samples. Using novel simulated datasets, which permit exact accuracy verification, XBS was compared to the UVP and MTBseq pipelines. Accuracy statistics showed that all three pipelines performed equally well for sequence data that resemble those obtained from culture isolates of high depth of coverage and low-level contamination. In the complex genomic regions, however, XBS accurately identified 9.0 % more SNPs and 8.1 % more single nucleotide insertions and deletions than the WHO-endorsed unified analysis variant pipeline. XBS also had superior accuracy for sequence data that resemble those obtained directly from sputum samples, where depth of coverage is typically very low and contamination levels are high. XBS was the only pipeline not affected by low depth of coverage (5–10×), type of contamination and excessive contamination levels (>50 %). Simulation results were confirmed using whole genome sequencing (WGS) data from clinical samples, confirming the superior performance of XBS with a higher sensitivity (98.8%) when analysing culture isolates and identification of 13.9 % more variable sites in WGS data from sputum samples as compared to MTBseq, without evidence for false positive variants when rRNA regions were excluded. The XBS pipeline facilitates sequencing of less-than-perfect Mtb samples. These advances will benefit future clinical applications of Mtb sequencing, especially WGS directly from clinical specimens, thereby avoiding in vitro biases and making many more samples available for drug resistance and other genomic analyses. The additional genetic resolution and increased sample success rate will improve genome-wide association studies and sequence-based transmission studies.
A species-wide genetic atlas of antimicrobial resistance in Clostridioides difficile
Authors: Imwattana Korakrit , Rodríguez César , Riley Thomas V. , Knight Daniel R.
Published: November 2021

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of

Clostridioides difficile

infection (CDI), the leading healthcare-related gastrointestinal infection in the world. An association between AMR and CDI outbreaks is well documented, however, data is limited to a few ‘epidemic’ strains in specific geographical regions. Here, through detailed analysis of 10 330 publicly-available

C. difficile

genomes from strains isolated worldwide (spanning 270 multilocus sequence types (STs) across all known evolutionary clades), this study provides the first species-wide snapshot of AMR genomic epidemiology in

C. difficile

. Of the 10 330

C

.

difficile

genomes, 4532 (43.9 %) in 89 STs across clades 1–5 carried at least one genotypic AMR determinant, with 901 genomes (8.7 %) carrying AMR determinants for three or more antimicrobial classes (multidrug-resistant, MDR). No AMR genotype was identified in any strains belonging to the cryptic clades.

C. difficile

from Australia/New Zealand had the lowest AMR prevalence compared to strains from Asia, Europe and North America (P<0.0001). Based on the phylogenetic clade, AMR prevalence was higher in clades 2 (84.3 %), 4 (81.5 %) and 5 (64.8 %) compared to other clades (collectively 26.9 %) (P<0.0001). MDR prevalence was highest in clade 4 (61.6 %) which was over three times higher than in clade 2, the clade with the second-highest MDR prevalence (18.3 %). There was a strong association between specific AMR determinants and three major epidemic

C. difficile

STs: ST1 (clade 2) with fluoroquinolone resistance (mainly T82I substitution in GyrA) (P<0.0001), ST11 (clade 5) with tetracycline resistance (various tet-family genes) (P<0.0001) and ST37 (clade 4) with macrolide-lincosamide-streptogramin B (MLSB) resistance (mainly ermB) (P<0.0001) and MDR (P<0.0001). A novel and previously overlooked tetM-positive transposon designated Tn6944 was identified, predominantly among clade 2 strains. This study provides a comprehensive review of AMR in the global

C. difficile

population which may aid in the early detection of drug-resistant

C. difficile

strains, and prevention of their dissemination worldwide.
Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa
Authors: Cornick Jennifer , Musicha Patrick , Peno Chikondi , Seager Ezgi , Iroh Tam Pui-Ying , Bilima Sithembile , Bennett Aisleen , Kennedy Neil , Feasey Nicholas , Heinz Eva , Cain Amy K.
Published: November 2021

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant

Klebsiella pneumoniae

was investigated using genomic characterisation. All

K. pneumoniae

bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of

K. pneumoniae

BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.
On biological flow networks: Antagonism between hydrodynamic and metabolic stimuli as driver of topological transitions
Authors: Kramer Felix , Modes Carl D.
Published: November 2021

A plethora of computational models have been developed in recent decades to account for the morphogenesis of complex biological fluid networks, such as capillary beds. Contemporary adaptation models are based on optimization schemes where networks react and adapt toward given flow patterns. Doing so, a system reduces dissipation and network volume, thereby altering its final form. Yet, recent numeric studies on network morphogenesis, incorporating uptake of metabolites by the embedding tissue, have indicated the conventional approach to be insufficient. Here, we systematically study a hybrid-model which combines the network adaptation schemes intended to generate space-filling perfusion as well as optimal filtration of metabolites. As a result, we find hydrodynamic stimuli (wall-shear stress) and filtration based stimuli (uptake of metabolites) to be antagonistic as hydrodynamically optimized systems have suboptimal uptake qualities and vice versa. We show that a switch between different optimization regimes is typically accompanied with a complex transition between topologically redundant meshes and spanning trees. Depending on the metabolite demand and uptake capabilities of the adaptating networks, we are further able to demonstrate the existence of nullity re-entrant behavior and the development of compromised phenotypes such as dangling non-perfused vessels and bottlenecks.

Author summary

Biological flow networks, such as capillaries, do not grow fully developed and matured in their final and functional form. Instead they grow a rudimentary network which self-organizes bit by bit in the context of their surrounding tissue, perfusion and other stimuli. Interestingly, it has been repeatedly shown that this development is mechano-transductional in nature, coupling complex bio-chemical signaling to mechanical cues such as wall-shear stress. In accordance with previous studies we propose a minimal hybrid model that takes into account stimuli in the form of the actual metabolite uptake of the surrounding tissue and the conventional wall-shear stress approach, and incorporate these into the metabolic cost function scheme. We present a numeric evaluation of our model, displaying the antagonistic interplay of uptake and shear stress driven morphogenesis as well as the topological ramifications and frustrated network formations, i.e. dangling branches, bottlenecks and re-entrant behavior in terms of redundancy transitions.
Cell cycle-dependent chromatin dynamics at replication origins
Authors: Li Yulong , Hartemink Alexander J. , MacAlpine David M.
Published: November 2021

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell cycle dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S-phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between the chromatin occupancy at the ACS and origin efficiency occurred in early S-phase consistent with the rate limiting formation of the Cdc45-Mcm2-7-GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S-phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell cycle-regulated chromatin dynamics and how they relate to the regulation of origin activity.
Rapid nuclear deadenylation of mammalian messenger RNA
Authors: Alles Jonathan , Legnini Ivano , Pacelli Maddalena , Rajewsky Nikolaus
Published: November 2021

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition, and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened within minutes after transcription. Our data show that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs escape rapid nuclear deadenylation. Modelling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggest that nuclear deadenylation is a key mechanism for regulating mRNA stability, abundance, and subcellular localization.
The Delta variant of SARS-CoV-2 maintains high sensitivity to interferons in human lung cells
Authors: Nchioua Rayhane , Schundner Annika , Klute Susanne , Noettger Sabrina , Zech Fabian , Koepke Lennart , Graf Alexander , Krebs Stefan , Blum Helmut , Kmiec Dorota , Frick Manfred , Kirchhoff Frank , Sparrer Konstantin M.J.
Published: November 2021

Interferons are a major part of the anti-viral innate defense system. Successful pathogens, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), need to overcome these defenses to establish an infection. Early induction of interferons (IFNs) protects against severe coronavirus disease 2019 (COVID-19). In line with this, SARS-CoV-2 is inhibited by IFNs in vitro, and IFN-based therapies against COVID-19 are investigated in clinical trials. However, SARS-CoV-2 continues to adapt to the human population resulting in the emergence of variants characterized by increased transmission fitness and/or decreased sensitivity to preventive or therapeutic measures. It has been suggested that the efficient spread of these so-called “Variants of Concern” (VOCs) may also involve reduced sensitivity to IFNs. Here, we examined whether the four current VOCs (Alpha, Beta, Gamma and Delta) differ in replication efficiency or IFN sensitivity from an early isolate of SARS-CoV-2. All viruses replicated in a human lung cell line and in iPSC-derived alveolar type II cells (iAT2). The Delta variant showed accelerated replication kinetics and higher infectious virus production compared to the early 2020 isolate. Replication of all SARS-CoV-2 VOCs was reduced in the presence of exogenous type I, II and III IFNs. On average, the Alpha variant was the least susceptible to IFNs and the Alpha, Beta and Gamma variants show increased resistance against type III IFN. Although the Delta variant has outcompeted all other variants in humans it remained as sensitive to IFNs as an early 2020 SARS-CoV-2 isolate. This suggests that increased replication fitness rather than IFN resistance may be a reason for its dominance. Our results may help to understand changes in innate immune susceptibility of VOCs, and inform clinical trials exploring IFN-based COVID-19 therapies.
Persistence of Plasmodium cynomolgi hypnozoites in cynomolgus monkey iPS-derived hepatocytes
Authors: Pellisson Mélanie , Zeeman Anne-Marie , Doll Thierry , Kirchhofer-Allan Lucy , Schuierer Sven , Roma Guglielmo , Flannery Erika L. , Mikolajczak Sebastian A. , Kocken Clemens H. M. , Maeser Pascal , Rottmann Matthias , Mueller Matthias
Published: November 2021

Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.
Global Mutational Sweep of SARS-CoV-2: from Chaos to Order
Authors: Wang Xin , Hu Mingda , Jin Yuan , Wang Boqian , Zhao Yunxiang , Liang Long , Yue Junjie , Ren Hongguang
Published: November 2021

Analysis of large-scale genome sequences demonstrates the mutation of SARS-CoV-2 has been undergoing significant sweeps. Driven by emerging variants, global sweeps are accelerated and purified over time. This may prolong the pandemic with repeating epidemics, presenting challenges to the control and prevention of SARS-CoV-2.
Mitogen-activated protein kinases associated sites of tobacco REPRESSION OF SHOOT GROWTH regulates its localization in plant cells
Authors: Wang Luyao , Gui Ying , Yang Bingye , Si Fangjie , Guo Jianhua , Jiang Chunhao
Published: November 2021

Plant defense and growth rely on multiple transcriptional factors (TFs). REPRESSION OF SHOOT GROWTH (RSG) is known as one of the important TFs in tobacco (Nicotiana tabacum) with a basic leucine zipper domain. RSG was involved in plant gibberellin feedback regulation by inducing the expression of key genes. The tobacco calcium-dependent protein kinase, CDPK1 was reported to interact with RSG and manipulate its intracellular localization by phosphorylating Ser-114 of RSG. Here, we identified tobacco mitogen-activated protein kinase 3 (NtMPK3) as a RSG interacted protein kinase. Mutation of predicted MAPK-associated phosphorylation site of RSG (Thr-30, Ser-74 and Thr-135) significantly altered the intracellular localization of NtMPK3-RSG interaction complex. Nuclear transport of RSG and its amino acids mutants (T30A and S74A) were observed after treated with plant defense elicitor peptide flg22 in 5 min, while the two mutated RSG swiftly relocalized in tobacco cytoplasm in 30 min. Moreover, triple points mutation of RSG (T30A/S74A/T135A) mimics constant unphosphorylated status, and predominantly localized in tobacco cytoplasm. RSG (T30A/S74A/T135A) showed no relocalization effect under the treatments of either flg22, B. cereus AR156 or GA3, and was impaired in its role as TFs. Our results suggest that MAPK associated phosphorylation sites of RSG regulate its localization in tobacco and constant unphosphorylation of RSG in Thr-30, Ser-74 and Thr-135 keeps RSG predominantly localized in cytoplasm.
Esrra regulates Rplp1-mediated translation of lysosome proteins suppressed in non-alcoholic steatohepatitis and reversed by alternate day fasting
Authors: Tripathi Madhulika , Gauthier Karine , Sandireddy Reddemma , Zhou Jin , Gupta Priyanka , Sakthivel Suganya , Teo Wei Wen , Naing Yadanar Than , Arul Kabilesh , Tikno Keziah , Park Sung-Hee , Wu Yajun , Wang Lijin , Bay Boon-Huat , Sun Lei , Giguere Vincent , Chow Pierce K. H. , Ghosh Sujoy , McDonnell Donald P. , Yen Paul M. , Singh Brijesh K.
Published: November 2021

Background

Currently, little is known about the mechanism(s) regulating global and specific protein translation during non-alcoholic steatohepatitis (NASH).

Methods

We used puromycin-labelling, polysome profiling, ChIPseq and ChIP-qPCR, and gene manipulationin vitroand in dietary mouse models of NASH in this study.

Results

Using unbiased label-free quantitative proteome, puromycin-labelling and polysome profiling, we observed a global decrease in protein translation during lipotoxicity in human primary hepatocytes, mouse hepatic AML12 cells, and livers from a dietary mouse model of NASH. Interestingly, proteomic analysis showed that Rplp1, which regulates ribosome and translation pathways, was one of the most downregulated proteins. Moreover, decreased Esrra expression and binding to the Rplp1 promoter, diminished Rplp1 gene expression during lipotoxicity. This, in turn, reduced global protein translation and Esrra/Rplp1-dependent translation of lysosome (Lamp2, Ctsd) and autophagy (sqstm1, Map1lc3b) proteins. Of note, Esrra did not increase its binding to these gene promoters or their gene transcription, confirming its regulation of their translation during lipotoxicity. Notably, hepatic Esrra-Rplp1-dependent translation of lysosomal and autophagy proteins also was impaired in NASH patients and liver-specificEsrraknockout mice.

Remarkably, alternate day fasting induced Essra-Rplp1-dependent expression of lysosomal proteins, restored autophagy, and reduced lipotoxicity, inflammation, and fibrosis in hepatic cell culture andin vivomodels of NASH.

Conclusion

Esrra regulation of Rplp1-mediated translation of lysosome / autolysosome proteins was downregulated during NASH. Alternate day fasting activated this novel pathway and improved NASH, suggesting that Esrra and Rplp1 may serve as therapeutic targets for NASH. Our findings also provided the first example of a nuclear hormone receptor, Esrra, to not only regulate transcription but also protein translation, via induction of Rplp1.
Genome-wide identification of novel sRNAs in Streptococcus mutans
Authors: Krieger Madeline C , Merritt Justin , Raghavan Rahul
Published: November 2021

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, non-coding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA, SmsR4. Our data demonstrate that SmsR4 regulates the EIIA component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria.

IMPORTANCE

Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.
Membrane adaptation in the hyperthermophilic archaeon Pyrococcus furiosus relies upon a novel strategy involving glycerol monoalkyl glycerol tetraether lipids
Authors: Tourte Maxime , Schaeffer Philippe , Grossi Vincent , Oger Philippe M.
Published: November 2021

Microbes preserve membrane functionality under fluctuating environmental conditions by modulating their membrane lipid composition. Although several studies have documented membrane adaptations in Archaea, the influence of most biotic and abiotic factors on archaeal lipid compositions remains underexplored. Here, we studied the influence of temperature, pH, salinity, the presence/absence of elemental sulfur, the carbon source, and the genetic background on the core lipid composition of the hyperthermophilic neutrophilic marine archaeon Pyrococcus furiosus. Every growth parameter tested affected the core lipid composition to some extent, the carbon source and the genetic background having the greatest influence. Surprisingly, P. furiosus appeared to only marginally rely on the two major responses implemented by Archaea, i.e., the regulation of the ratio of diether to tetraether lipids and that of the number of cyclopentane rings in tetraethers. Instead, this species increased the ratio of glycerol monoalkyl glycerol tetraethers (GMGT, aka. H-shaped tetraethers) to glycerol dialkyl glycerol tetrathers (GDGT) in response to decreasing temperature and pH and increasing salinity, thus providing for the first time evidence of adaptive functions for GMGT. Besides P. furiosus, numerous other species synthesize significant proportions of GMGT, which suggests that this unprecedented adaptive strategy might be common in Archaea.

Significance statement

We describe here the membrane adaptive strategies the hyperthermophilic, neutrophilic, and marine model archaeon Pyrococcus furiosus implements in response to one of the largest sets of environmental stressors tested to date, including temperature, pH, salinity, presence/absence of elemental sulfur, carbon source, and genetic background. In contrast to the other archaea investigated so far, which response mainly involves the modulation of their diether/tetraether ratio and/or of their average number of cyclopentane rings, P. furiosus regulates its monoalkyl (so called H-shaped) to dialkyl tetraether ratio. Our study thus provides for the first time evidence of adaptive functions of archaeal monoalkyl tetraethers towards low temperature and pH and high salinity.
Recognition and deubiquitination of free 40S for translational reset by Otu2
Authors: Ikeuchi Ken , Ivic Nives , Cheng Jingdong , Buschauer Robert , Matsuo Yoshitaka , Fröhlich Thomas , Berninghausen Otto , Inada Toshifumi , Becker Thomas , Beckmann Roland
Published: November 2021

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.
Clathrin-mediated endocytosis-independent function of VAN3 ARF GTPase Activating Protein at the plasma membrane
Authors: Adamowski Maciek , Friml Jiří
Published: November 2021

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. TheArabidopsis thalianaARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally invan3-1knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.
The role of clathrin in exocytosis and the mutual regulation of endo- and exocytosis in plant cells
Authors: Adamowski Maciek , Matijević Ivana , Friml Jiří
Published: November 2021

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and thetrans-Golgi Network/Early Endosome (TGN/EE). While the role of clathrin as a major component of endocytosis at the PM is well established, its function at TGN/EE, possibly in exocytosis or the vacuolar pathway, is a matter of debate. This shared function of clathrin also opens a question whether plant cells possess a homeostatic mechanisms that balance rates of opposite trafficking routes, such as endo- and exocytosis. Here we address these questions using lines inducibly silencingCLATHRIN HEAVY CHAIN(CHC). We find a relocation of exocytic soluble and integral membrane protein cargoes to the vacuole, supporting a function of clathrin in exocytosis. A comparison with lines overexpressing AUXILIN-LIKE1, where inhibition of CME precedes rerouting of secretory cargoes, does not support a homeostatic regulatory mechanism adjusting exocytosis to the rates of endocytosis. Complementary experiments reveal only minor and variably detectable reductions in the rates of CME in secretory mutants, also not indicative of a converse homeostatic mechanism adjusting rates of endocytosis to the rates of secretion.
Among retroviral integrases prototype foamy virus integrase displays unique biochemical activities
Authors: Rabe Anthony J. , Tan Yow Yong , Larue Ross C. , Yoder Kristine E.
Published: November 2021

Integrase enzymes of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 seconds, PFV intasomes do not commit to target DNA during their reaction lifetime. Together these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.
In vitro culture of bovine fibroblasts using select serum-free media supplemented withChlorella vulgarisextract
Authors: Defendi-Cho Galileo , Gould Timothy M.
Published: November 2021

Standard cell culture practices require addition of animal-derived serum to culture media to achieve adequate cell growth. Typically, 5-10% by volume of fetal bovine serum (FBS) is used, which accounts for a vast majority of the cost of media while also imposing environmental and ethical concerns associated with the use of animal serum. Here we tested the efficacy of culturing cells by replacing serum in the media with algae extract and select additives. Using LC-MS, we compared molecular signatures of FBS toChlorellaalgae extracts and identified NAD(H)/NADP(H) as common and relatively abundant features in their characteristic profiles. Bovine fibroblasts, cultured in serum-free media supplemented withC. vulgarisextract and just two growth factors plus insulin, showed significant growth with enhanced viability compared to control cells cultured without serum, albeit still lower than that of controls cultured with 10% FBS. Moreover,C. vulgarisextract enhanced cell viability beyond that of cells cultured with the two growth factors and insulin alone. These results suggest that key components in serum which are essential for cell growth may also be present inC. vulgarisextract, demonstrating that it may be used at least as a partial alternative to serum for cell culture applications.
Allelic Variation in Gene Expression Is Common in the Human Genome
Authors: Lo H. Shuen , Wang Zhining , Hu Ying , Yang Howard H. , Gere Sheryl , Buetow Kenneth H. , Lee Maxwell P.
Published: November 2021

Variations in gene sequence and expression underlie much of human variability. Despite the known biological roles of differential allelic gene expression resulting from X-chromosome inactivation and genomic imprinting, a large-scale analysis of allelic gene expression in human is lacking. We examined allele-specific gene expression of 1063 transcribed single-nucleotide polymorphisms (SNPs) by using Affymetrix HuSNP oligo arrays. Among the 602 genes that were heterozygous and expressed in kidney or liver tissues from seven individuals, 326 (54%) showed preferential expression of one allele in at least one individual, and 170 of those showed greater than fourfold difference between the two alleles. The allelic variation has been confirmed by real-time quantitative PCR experiments. Some of these 170 genes are known to be imprinted, such as SNRPN, IPW, HTR2A, and PEG3. Most of the differentially expressed genes are not in known imprinting domains but instead are distributed throughout the genome. Our studies demonstrate that variation of gene expression between alleles is common, and this variation may contribute to human variability.
Comparative DNA Sequence Analysis of Wheat and Rice Genomes
Authors: Sorrells Mark E. , La Rota Mauricio , Bermudez-Kandianis Catherine E. , Greene Robert A. , Kantety Ramesh , Munkvold Jesse D. , Miftahudin  , Mahmoud Ahmed , Ma Xuefeng , Gustafson Perry J. , Qi Lili L. , Echalier Benjamin , Gill Bikram S. , Matthews David E. , Lazo Gerard R. , Chao Shiaoman , Anderson Olin D. , Edwards Hugh , Linkiewicz Anna M. , Dubcovsky Jorge , Akhunov Eduard D. , Dvorak Jan , Zhang Deshui , Nguyen Henry T. , Peng Junhua , Lapitan Nora L.V. , Gonzalez-Hernandez Jose L. , Anderson James A. , Hossain Khwaja , Kalavacharla Venu , Kianian Shahryar F. , Choi Dong-Woog , Close Timothy J. , Dilbirligi Muharrem , Gill Kulvinder S. , Steber Camille , Walker-Simmons Mary K. , McGuire Patrick E. , Qualset Calvin O.
Published: November 2021

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.
A Gene Recommender Algorithm to Identify Coexpressed Genes inC. elegans
Authors: Owen Art B. , Stuart Josh , Mach Kathy , Villeneuve Anne M. , Kim Stuart
Published: November 2021

One of the most important uses of whole-genome expression data is for the discovery of new genes with similar function to a given list of genes (the query) already known to have closely related function. We have developed an algorithm, called the gene recommender, that ranks genes according to how strongly they correlate with a set of query genes in those experiments for which the query genes are most strongly coregulated. We used the gene recommender to find other genes coexpressed with several sets of query genes, including genes known to function in the retinoblastoma complex. Genetic experiments confirmed that one gene (JC8.6) identified by the gene recommender acts withlin-35Rb to regulate vulval cell fates, and that another gene (wrm-1) acts antagonistically. We find that the gene recommender returns lists of genes with better precision, for fixed levels of recall, than lists generated using theC. elegansexpression topomap.
A Large Database of Chicken Bursal ESTs as a Resource for the Analysis of Vertebrate Gene Function
Authors: Abdrakhmanov Igor , Lodygin Dmitry , Geroth Paul , Arakawa Hiroshi , Law Andy , Plachy Jiri , Korn Berndt , Buerstedde Jean-Marie
Published: November 2021

Chicken B cells create their immunoglobulin repertoire within the Bursa of Fabricius by gene conversion. The high homologous recombination activity is shared by the bursal B-cell-derived DT40 cell line, which integrates transfected DNA constructs at high rates into its endogenous loci. Targeted integration in DT40 is used frequently to analyze the function of genes by gene disruption. In this paper, we describe a large database of >7000 expressed sequence tags (ESTs) from bursal lymphocytes that should be a valuable resource for the identification of gene disruption targets in DT40. ESTs of interest can be recognized easily by online BLAST or keyword searches. Because the database reflects the gene expression profile of bursal lymphocytes, it provides valuable hints as to which genes might be involved in B-cell–specific processes related to immunoglobulin repertoire formation, signal transduction, transcription, and apoptosis. This large collection of chicken ESTs will also be useful for gene expression studies and comparative gene mapping within the chicken genome project. Details of the bursal EST sequencing project and access to database search forms can be found on the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html).

[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AJ392050–AJ399459.]