bluuebunny/crossref_metadata_2025 huggingface dataset:
ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver
Authors: Kurosawa Kiamu , Nakano Masataka , Yokoseki Itsuki , Nagaoka Mai , Takemoto Seiya , Sakai Yoshiyuki , Kobayashi Kaoru , Kazuki Yasuhiro , Fukami Tatsuki , Nakajima Miki
Published: February 2023

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug□drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXRin vitroandin vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to theCYP3A4promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.
Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice
Authors: Borin Joshua M. , Liu Roland , Wang Yanhan , Wu Tsung-Chin , Chopyk Jessica , Huang Lina , Kuo Peiting , Ghose Chandrabali , Meyer Justin R. , Tu Xin M. , Schnabl Bernd , Pride David T.
Published: February 2023

Background

The gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mice’s gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss.

Results

We gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes.

Conclusions

This study confirms that virome-mediated perturbations can alter the fecal microbiome in anin vivomodel and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.
Somatic nuclear mitochondrial DNA insertions are prevalent in the human brain and accumulate over time in fibroblasts
Authors: Zhou Weichen , Karan Kalpita R. , Gu Wenjin , Klein Hans-Ulrich , Sturm Gabriel , De Jager Philip L. , Bennett David A. , Hirano Michio , Picard Martin , Mills Ryan E
Published: February 2023

The transfer of mitochondrial DNA into the nuclear genomes of eukaryotes (Numts) has been linked to lifespan in non-human species1–3and recently demonstrated to occur in rare instances from one human generation to the next4. Here we investigated numtogenesis dynamics in humans in two ways. First, we quantified Numts in 1,187 post-mortem brain and blood samples from different individuals. Compared to circulating immune cells (n=389), post-mitotic brain tissue (n=798) contained more Numts, consistent with their potential somatic accumulation. Within brain samples we observed a 5.5-fold enrichment of somatic Numt insertions in the dorsolateral prefrontal cortex compared to cerebellum samples, suggesting that brain Numts arose spontaneously during development or across the lifespan. Moreover, more brain Numts was linked to earlier mortality. The brains of individuals with no cognitive impairment who died at younger ages carried approximately 2 more Numts per decade of life lost than those who lived longer. Second, we tested the dynamic transfer of Numts using a repeated-measures WGS design in a human fibroblast model that recapitulates several molecular hallmarks of aging5. These longitudinal experiments revealed a gradual accumulation of one Numt every ∼13 days. Numtogenesis was independent of large-scale genomic instability and unlikely driven cell clonality. Targeted pharmacological perturbations including chronic glucocorticoid signaling or impairing mitochondrial oxidative phosphorylation (OxPhos) only modestly increased the rate of numtogenesis, whereas patient-derivedSURF1-mutant cells exhibiting mtDNA instability accumulated Numts 4.7- fold faster than healthy donors. Combined, our data document spontaneous numtogenesis in human cells and demonstrate an association between brain cortical somatic Numts and human lifespan. These findings open the possibility that mito-nuclear horizontal gene transfer among human post-mitotic tissues produce functionally-relevant human Numts over timescales shorter than previously assumed.
High-level ceftazidime-avibactam resistance inEscherichia coliconferred by the novel plasmid-mediated beta-lactamase CMY-185 variant
Authors: Shropshire William C. , Endres Bradley T. , Borjan Jovan , Aitken Samuel L. , Bachman William C. , McElheny Christi L. , Khan Ayesha , Bhatti Micah M. , Saharasbhojane Pranoti , Kawai Akito , Shields Ryan K. , Shelburne Samuel A. , Doi Yohei
Published: February 2023

Objectives

To characterize ablaCMYvariant associated with ceftazidime-avibactam (CZA) resistance from a serially collectedEscherichia coliisolate.

Methods

A patient with an intra-abdominal infection due to recurrentE. coliwas treated with CZA. On day 48 of CZA therapy,E. coliwith a CZA MIC of >256 mg/L was identified from abdominal drainage. Illumina WGS was performed on all isolates to identify potential resistance mechanisms. Site-directed mutants of CMY β-lactamase were constructed to identify amino acid residues responsible for CZA resistance.

Results

WGS revealed that all three isolates wereE. coliST410. The CZA-resistant strain uniquely acquired a novel CMY β-lactamase gene, herein calledblaCMY-185, harbored on an IncIγ-type conjugative plasmid. The CMY-185 enzyme possessed four amino acid substitutions relative to CMY-2 including A114E, Q120K, V211S, and N346Y and conferred high-level CZA resistance with an MIC of 32 mg/L. Single CMY-2 mutants did not confer reduced CZA susceptibility. However, double and triple mutants containing N346Y previously associated with CZA resistance in other AmpC enzymes, conferred CZA MICs ranging between 4 and 32 mg/L as well as reduced susceptibility to the newly developed cephalosporin, cefiderocol. Molecular modelling suggested that the N346Y substitution confers the reduction of avibactam inhibition due to the steric hindrance between the side chain of Y346 and the sulfate group of avibactam.

Conclusion

We identified CZA resistance inE. coliassociated with a novel CMY variant. Unlike other AmpC enzymes, CMY-185 appears to require an additional substitution on top of N346Y to confer CZA resistance.
Conditioning electrical stimulation fails to enhance sympathetic axon regeneration
Authors: Tian Tina , Patel Kevin , Kim David , SiMa HaoMin , Harris Alandrea R. , Owyoung Jordan N. , Ward Patricia J.
Published: February 2023

Peripheral nerve injuries are common, and there is a critical need for the development of novel treatments to complement surgical repair. Conditioning electrical stimulation (CES) is a novel variation of the well-studied perioperative electrical stimulation treatment paradigm. CES is a clinically attractive alternative because of its ability to be performed at the bedside prior to a scheduled nerve repair surgery. Although 60 minutes of CES has been shown to enhance motor and sensory axon regeneration, the effects of CES on sympathetic regeneration are unknown. We investigated how two clinically relevant CES paradigms (10 minutes and 60 minutes) impact sympathetic axon regeneration and distal target reinnervation. Our results indicate that the growth of sympathetic axons is inhibited by CES at acute time points, and at a longer survival time point post-injury, there is no difference between sham CES and the CES groups.

We conclude sympathetic axons may retain some regenerative ability, but no enhancement is exhibited after CES, which may be accounted for by the inability of the electrical stimulation paradigm to recruit the small-caliber sympathetic axons into activity. Furthermore, 10-minute CES did not enhance motor and sensory regeneration with a direct repair, and neither 60-minute nor 10-minute CES enhanced motor and sensory regeneration through a graft. Further studies will be needed to optimize electrical stimulation parameters to enhance the regeneration of all neuron types.
Targeting fungal BET bromodomains as apan-Candidaantifungal strategy
Authors: Wei Kaiyao , Overhulse Justin M. , Arlotto Marie , Zhou Yingsheng , Dupper Nathan J. , Kashemirov Boris A. , Garnaud Cécile , Bourgine Gaëlle , Cornet Muriel , Rabut Gwenaël , McKenna Charles E. , Petosa Carlo , Govin Jérôme
Published: February 2023

Small molecules that target one or both bromodomains (BDs) of human BET proteins are intensely studied as potential new therapeutics against cancer, diabetes and other diseases. The BDs of the fungal BET protein Bdf1 are essential for the human fungal pathogenCandida albicans, suggesting BET inhibition as a potential antifungal strategy. However, while the inactivation of both Bdf1 BDs is lethal, that of a single BD only modestly affects viability, implying the need to develop antifungal compounds that selectively target both Bdf1 BDs without inhibiting human BDs. Here, we investigate Bdf1 as a potential antifungal target inCandida glabrata, an invasiveCandidaspecies phylogenetically distant fromC. albicansand of increasing medical concern. We show that Bdf1 BD functionality is essential inC. glabrataand identify a phenyltriazine derivative that targets both Bdf1 BDs with selectivity over human BET BDs. We show that human BET BDs can functionally replace Bdf1 BDs inC. glabrataand we use the humanized strains to demonstrate on-target antifungal activity of the phenyltriazine compound. Moreover, by exploiting the humanized and parental fungal strains we identified BET inhibitor I-BET726 to have potent antifungal activity against a broad spectrum ofCandidaspecies, including azole- and echinocandin-resistant clinicalC. albicansandC. glabrataisolates. Crystal structures suggest how to improve the potency and selectivity of these compounds. Taken together, our findings provide compelling support for the development of BET inhibitors as potential pan-Candidaantifungal therapeutics.
Too hot or too disturbed? Temperatures more than hikers affect circadian activity of females in northern chamois
Authors: Thel Lucie , Garel Mathieu , Marchand Pascal , Bourgoin Gilles , Loison Anne
Published: February 2023

Recreational activities often result in a spatial and/or temporal activity shift in wildlife. With the concurrent development of outdoor activities and increase in temperatures due to climate change, mountain species face increasing pressures in terms of managing their activity pattern to limit both risk exposure and thermal discomfort. Using more than 15 years of long-term GPS and activity sensor data, we investigated how female northern chamois,Rupicapra rupicapra rupicapra, adjust their summer circadian activity to spatiotemporal variation in both temperatures and hikers’ presence. Chamois behaviour was more affected by high temperatures than by hikers’ presence. During the hottest days, they shifted their activity peak earlier in the morning, were more active at night, less active during daytime and had longer morning and evening peaks compared to the coldest days. Global activity was only slightly different during the hottest than the coldest days. Conversely, hikers’ disturbance had weak effects on activity levels and on the timing of activity peaks. This is especially true for temporal disturbance (weekdays versus weekends and public holidays), possibly because most weekdays in summer fell during school holidays. During the hottest conditions, the morning activity peak was shorter and the evening peak longer in females living in the most exposed areas compared to females living in the least exposed areas. One possible explanation for the overall low effect of hikers’ disturbance may be that behavioural changes buffering animals from high temperatures and hikers’ presence (e.g. moving away from trails) allow them to just marginally modify their activity pattern. In the context of ongoing socioenvironmental changes, it is critical to conserve habitats providing thermal refuges against summer heat and protection from disturbance to mitigate potential detrimental consequences.
A panel of TDP-43-regulated splicing events verify loss of TDP-43 function in amyotrophic lateral sclerosis brain tissue
Authors: Cao Maize C. , Ryan Brigid , Wu Jane , Curtis Maurice , Faull Richard , Dragunow Mike , Scotter Emma L.
Published: February 2023

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and exon changes are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depletingTARDBPfrom post-mortem human brain pericytes, a manipulablein vitroprimary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database (https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a ‘differential exon usage panel’ with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons and changed exon usage discriminate ALS brain tissue from controls, verifying TDP-43 loss of function as a pathomechanism in ALS. We propose that TDP-43-regulated splicing markers with most predictive value for therapeutic intervention will be those based on splicing events that occur both in tissues/biofluids amenable to sampling, and in brain tissue susceptible to disease.
Imaging Microglia Surveillance during Sleep-wake Cycles in Freely Behaving Mice
Authors: Gu Xiaochun , Zhao Zhong , Chen Xueli , Zhang Lifeng , Fang Huaqiang , Zhao Ting , Ju Shenghong , Gao Weizheng , Qian Xiaoyu , Wang Xianhua , Zhang Jue , Cheng Heping
Published: February 2023

Microglia surveillance manifests itself as dynamic changes in cell morphology and functional remodeling. Whether and how microglia surveillance is coupled to brain state switches during natural sleep-wake cycles remain unclear. To address this question, we used miniature two-photon microscopy (mTPM) to acquire time-lapse high-resolution microglia images of the somatosensory cortex, along with EEG/EMG recordings and behavioral video, in freely-behaving mice. We uncovered fast and robust brain state-dependent changes in microglia surveillance, occurring in parallel with sleep dynamics and early-onset phagocytic microglial contraction during sleep deprivation stress. We also detected local norepinephrine fluctuation occurring in a sleep state-dependent manner. We showed that the locus coeruleus-norepinephrine system, which is crucial to sleep homeostasis, is required for both sleep state-dependent and stress-induced microglial responses and β2-adrenergic receptor signaling plays a significant role in this process. These results provide direct evidence that microglial surveillance is exquisitely tuned to signals and stressors that regulate sleep dynamics and homeostasis so as to adjust its varied roles to complement those of neurons in the brain.In vivoimaging with mTPM in freely behaving animals, as demonstrated here, opens a new avenue for future investigation of microglia dynamics and sleep biology in freely behaving animals.
Single molecule DNA methylation and hydroxymethylation reveal unique epigenetic identity profiles of T helper cells
Authors: Goldsmith Chloe , Fesneau Olivier , Thevin Valentin , Matias Maria I. , Perrault Julie , Abid Ali Hani , Taylor Naomi , Dardalhon Valérie , Marie Julien C. , Hernandez-Vargas Hector
Published: February 2023

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation (5mC) and cytosine hydroxymethylation (5hmC) are DNA modifications that identify stable cell phenotypes but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a new method to profile Th cell identity using cas9-targeted single molecule nanopore sequencing and found that 5mC and 5hmC can be used as markers of cellular identity. Targeting as few as 10 selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mC/5hmC patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our analysis demonstrates the importance of epigenetic regulation by 5mC and 5hmC modifications in the establishment of Th cell identity. Furthermore, our data highlight the potential to exploit this immune profiling application to elucidate the pathogenic role of Th transition states in autoimmune diseases.
FarmGTEx TWAS-server: an interactive web server for customized TWAS analysis in both human and farm animals
Authors: Zhang Zhenyang , Chen Zitao , Teng Jinyan , Liu Shuli , Lin Qing , Gao Yahui , Bai Zhonghao ,   The FarmGTEx Consortium, Li Bingjie , Liu George , Zhang Zhe , Pan Yuchun , Zhang Zhe , Fang Lingzhao , Wang Qishan
Published: February 2023

Transcriptome-wide association study (TWAS) is a powerful strategy for elucidating the molecular mechanisms behind the genetic loci of complex phenotypes. However, TWAS analysis is still daunting in many species due to the complication of the TWAS analysis pipeline, including the construction of the gene expression reference panel, gene expression prediction, and the subsequent association analysis in the large cohorts of genome-wide association study (GWAS). Farm animals are major protein sources and biomedical models for humans. To facilitate the translation of genetic findings across species, here we provide an interactive and easy-to-use multi-species TWAS web server for the entire community, called the FarmGTEx TWAS-server (http://twas.farmgtex.org), which is based on the GTEx and FarmGTEx projects. It includes gene expression data from 49, 34, and 23 tissues in 838 humans, 5,457 pigs, and 4,889 cattle, representing 38,180, 21,037, and 17,942 distinct eGenes in prediction models for humans, pigs, and cattle, respectively. It allows users to conduct gene expression prediction for any individuals with genotypes, GWAS summary statistics imputation, customized TWAS, and popular downstream functional annotation. It also provides 479,203, 1,208, and 657 tissue-gene-trait association trios for the research community, representing 1,129 human traits, 41 cattle traits, and 11 pig traits. In summary, the FarmGTEx TWAS-server is a one-stop solution for performing TWAS analysis for researchers without programming skills in both human and farm animal research communities. It will be maintained and updated timely within the FarmGTEx project to facilitate gene mapping and phenotype prediction within and across species.
A Structural Classification of the Variant Surface Glycoproteins of the African Trypanosome
Authors: Đaković Sara , Zeelen Johan , Gkeka Anastasia , Chandra Monica , Foti Konstantina , van Straaten Monique , Vlachou Evi P. , Aresta-Branco Francisco , Verdi Joseph P. , Papavasiliou F. Nina , Stebbins C. Erec
Published: February 2023

Long-term immune evasion by the African trypanosome is achieved through repetitive cycles of surface protein replacement with antigenically distinct versions of the dense Variant Surface Glycoprotein (VSG) coat. Thousands of VSG genes and pseudo-genes exist in the parasite genome that, together with genetic recombination mechanisms, allow for essentially unlimited immune escape from the host’s adaptive immune system. The diversity space of the “VSGnome” at the protein level was thought to be limited to a few related folds whose structures were determined more than 30 years ago. However, recent progress has shown that the VSGs possess significantly more architectural variation than had been appreciated. Here we combine experimental X-ray crystallography with deep-learning structural prediction using Alphafold to produce models of hundreds of VSG proteins. We classify the VSGnome into groups based on protein architecture and oligomerization state, contextualize recent bioinformatics clustering schemes, and extensively map VSG-diversity space. We demonstrate that in addition to the structural variability and post-translational modifications observed thus far, VSGs are also characterized by variations in oligomerization state and possess inherent flexibility and alternative conformations, lending additional variability to what is exposed to the immune system. Finally, these additional experimental structures and the hundreds of Alphafold predictions confirm that the molecular surfaces of the VSGs remain distinct from variant to variant, supporting the hypothesis that protein surface diversity is central to the process of antigenic variation used by this organism during infection.
Role of acetoacetyl-CoA synthetase in glucose uptake by HepG2 cells
Authors: Hasegawa Shinya , Imai Masahiko , Takahashi Noriko
Published: February 2023

Accelerated glucose metabolism is a common feature of cancer cells and represents a potential therapeutic target. Recent studies have revealed that lipid metabolism is related to glucose me-tabolism, especially glucose uptake. Acetoacetyl-CoA synthetase (AACS) converts acetoacetate, a ketone body, to acetoacetyl-CoA, which is incorporated into cholesterol and fatty acids. The AACS gene is highly expressed in HepG2 cells but not in the human normal liver. These results suggest a correlation between AACS and glucose metabolism. Therefore, we examined the relationship between AACS and glucose uptake in hepatocellular carcinoma (HepG2) cells. The expression of AACS was significantly upregulated when HepG2 cells were exposed to high concentrations of glucose. Lentiviruses, which code for shRNA against AACS (shAACS), were used to determine whether AACS knockdown affects the glucose uptake in HepG2 cells. AACS knockdown signifi-cantly reduced glucose uptake and increased the concentration of ketone bodies in the media, and treatment with β-hydroxybutyrate, a ketone body, attenuated glucose uptake. Moreover, the knockdown of AACS, i.e., the increased concentration of the intracellular ketone body, slightly attenuated cell proliferation and significantly increased the sensitivity to the anticancer agent sorafenib. These results suggest that AACS plays an important role in glucose uptake and cell proliferation and that ketone bodies may compete with glucose as an important energy source for cancer cells.
Mechanistic Modeling Explains the Production Dynamics of Recombinant Adeno-Associated Virus with the Baculovirus Expression Vector System
Authors: Destro Francesco , Srinivasan Prasanna , Kanter Joshua M. , Neufeld Caleb , Wolfrum Jacqueline M. , Barone Paul W. , Springs Stacy L. , Sinskey Anthony J. , Cecchini Sylvain , Kotin Robert M. , Braatz Richard D.
Published: February 2023

The demand for recombinant adeno-associated virus (rAAV) for gene therapy is expected to soon exceed current manufacturing capabilities, considering the expanding number of approved products and of pre-clinical and clinical stage studies. Current rAAV manufacturing processes have less-than-desired yields and produce a significant amount of empty capsids. Recently, FDA approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS). The BEVS technology, based on an invertebrate cell line derived fromSpodoptera frugiperda, demonstrated scalable production of high volumetric titers of full capsids. In this work, we develop a mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV virion maturation in the BEVS. The predictions of the model show good agreement with experimental measurements reported in the literature on rAAV manufacturing in the BEVS, including for TwoBac, ThreeBac, and OneBac constructs. The model is successfully validated against measured concentrations of structural and non-structural protein components, and of vector genome. We carry out a model-based analysis of the process, to provide insights on potential bottlenecks that limit the formation of full capsids. The analysis suggests that vector genome amplification is the limiting step for rAAV production in TwoBac. In turn, vector genome amplification is limited by low Rep78 levels. For ThreeBac, low vector genome amplification dictated by Rep78 limitation appears even more severe than in TwoBac. Transgene expression in the insect cell during rAAV manufacturing is also found to negatively influence the final rAAV production yields.
Multimodal meta-analysis of fecal metagenomes reveals microbial single nucleotide variants as superior biomarkers for early detection of colorectal cancer
Authors: Gao Wenxing , Gao Xiang , Zhu Lixin , Gao Sheng , Sun Ruicong , Feng Zhongsheng , Wu Dingfeng , Liu Zhanju , Zhu Ruixin , Jiao Na
Published: February 2023

Microbial signatures show remarkable potentials in predicting colorectal cancer (CRC). This study aimed to evaluate the diagnostic powers of multimodal microbial signatures, multi-kingdom species, genes, and single-nucleotide variants (SNVs) for detecting pre-cancerous adenomas. We performed cross-cohort analyses on whole metagenome sequencing data of 750 samples via xMarkerFinder to identify adenoma-associated microbial multimodal signatures. Our data revealed that fungal species outperformed species from other kingdoms with an area under the ROC curve (AUC) of 0.71 in distinguishing adenomas from controls, while classifier based on SNVs of bacterial species displayed the strongest diagnostic capability (AUC=0.89). SNV biomarkers also exhibited outstanding performances in three independent validation cohorts (AUCs = 0.83, 0.82, and 0.76, respectively) and were highly specific to adenoma. In further support of the above results, functional analyses revealed more frequent inter-kingdom associations between bacteria and fungi, and abnormalities in quorum sensing, purine and butanoate metabolism in adenoma, which were further validated in a newly-recruited cohort via qRT-PCR. Therefore, these data extend our understanding of adenoma-associated multimodal alterations in the gut microbiome and provide a rationale of microbial SNVs for the early detection of CRC.
Protein Sequence Design by Entropy-based Iterative Refinement
Authors: Zhou Xinyi , Chen Guangyong , Ye Junjie , Wang Ercheng , Zhang Jun , Mao Cong , Li Zhanwei , Hao Jianye , Huang Xingxu , Tang Jin , Ann Heng Pheng
Published: February 2023

Inverse Protein Folding (IPF) is an important task of protein design, which aims to design sequences compatible with a given backbone structure. Despite the prosperous development of algorithms for this task, existing methods tend to leverage limited and noisy residue environment when generating sequences. In this paper, we develop an iterative sequence refinement pipeline, which can refine the sequence generated by existing sequence design models. It selects and retains reliable predictions based on the model’s confidence in predicted distributions, and decodes the residue type based on a partially visible environment. The proposed scheme can consistently improve the performance of a number of IPF models on several sequence design benchmarks, and increase sequence recovery of the SOTA model by up to 10%. We finally show that the proposed model can be applied to redesign Transposon-associated transposase B. 8 variants exhibit improved gene editing activity among the 20 variants we proposed. Our code and a demo of the refinement pipeline are provided in the online colab.
A network-based analysis of signal use during approach interactions across sexes in chacma baboons (Papio ursinus griseipes)
Authors: Muschinski Jana , Mielke Alexander , Carvalho Susana
Published: February 2023

Greetings in primates fulfil important functions including navigation of rank, maintenance of social relationships, and potentially establishing coalition partnerships.Papiomakes a particularly valuable study genus as baboons show variation in greeting, male-male cooperation, philopatry, and social system. However, baboon greeting research has largely focused on male-male interactions, with female approach behaviour neglected except in relation to friendships and grunting. Most if not all signals seen in male-male greetings are also present in approaches between other sex combinations. To understand these signals further, their use in all sex combinations should be explored. We investigated approaches between male and female adult chacma baboons (Papio ursinus griseipes), the only savannah baboon reportedly lacking male-male cooperation, recorded in Gorongosa National Park, Mozambique. We compared male-male greetings with those of other baboon species, identified network clusters of co-occurring signals, and compared male and female approaches more broadly. Male-male approaches were similar to those in other baboon species. We identified several predictable signal combinations, ear-flattening with lip-smacking being a particularly strong signal of benign intent across sexes. Further research comparing greeting across sex combinations and species will help disentangle links between risk, cooperation, and greeting behaviour.
ORFanes in mitochondrial genomes of marine polychaetePolydora
Authors: Selifanova Maria , Demianchenko Oleg , Noskova Elizaveta , Pitikov Egor , Skvortsov Denis , Drozd Jana , Vatolkina Nika , Apel Polina , Kolodyazhnaya Ekaterina , Ezhova Margarita A. , Tzetlin Alexander B. , Neretina Tatiana V. , Knorre Dmitry A.
Published: February 2023

Most characterised metazoan mitochondrial genomes are compact and encode a small set of proteins that are essential for oxidative phosphorylation. However, in rare cases, invertebrate taxa have additional open reading frames (ORFs) in their mtDNA sequences. Here, we sequenced and analysed the mitochondrial genome of a polychaete worm,Polydora cf. ciliata, part of whose life cycle takes place in low-oxygen conditions. In the mitogenome, we found three “ORFane” regions (1063, 427, and 519 bp) that have no resemblance to any standard metazoan mtDNA gene but lack stop codons in one of the reading frames. Similar regions are found in the mitochondrial genomes of three other Polydora species andBocardiella hamata. All five species share the same gene order in their mitogenomes, which differ from that of other known spionidae mitogenomes. By analysing the ORFane sequences, we found that they are under negative selection pressure, contain conservative regions, and harbour predicted transmembrane domains.The codon adaptation indices (CAIs) of the ORFan genes were in the same range of values as the CAI of conventional protein-coding genes in corresponding mitochondrial genomes. Together, this suggests that ORFanes encode functional proteins. We speculate that the ORFanes originated from the conventional mitochondrial protein-coding genes which were duplicated when the Polydora/Bocardiella species complex separated from the rest of the Spionidae.

Significance statement

Metazoan mitochondrial genomes usually contain a conservative set of genes and features. However, mitogenomes of some species contain ORFanes – putative protein-coding genes without clear homology with other known sequences. In this study, we analysed three ORFanes in mitochondria of species of the genera Polydora and Bocardiella, which were absent in all other representatives of Spionidae. To the best of our knowledge, ORFanes haven’t been described in Annelida before. Sequence analysis of the ORFanes suggests they contain conservative regions and are likely translated into functional proteins. Our study features an uncommon case where new protein-coding genes emerged in the mitochondrial genomes of metazoa.
Mammalian Milk Glycomes: Connecting the Dots between Evolutionary Conservation and Biosynthetic Pathways
Authors: Thomès Luc , Karlsson Viktoria , Lundstrøm Jon , Bojar Daniel
Published: February 2023

Milk oligosaccharides (MOs) are among the most abundant constituents of breast milk and are essential for health and development. Biosynthesized from monosaccharides into complex sequences, MOs differ considerably between taxonomic groups. Even human MO biosynthesis is insufficiently understood, hampering evolutionary and functional analyses. Using a comprehensive resource of all published MOs from >100 mammals, we develop a nonparametric pipeline for generating and analyzing MO biosynthetic networks, which readily generalizes to other glycan classes. We then use evolutionary relationships and inferred intermediates of these networks to discover (i) distributional glycome biases, (ii) biosynthetic restrictions, such as reaction path dependence, and (iii) conserved biosynthetic modules. This allows us to prune and pinpoint biosynthetic pathways despite missing information. Machine learning and network analysis cluster species by their milk glycome, identifying characteristic sequence relationships and evolutionary gains/losses of motifs, MOs, and biosynthetic modules. These resources and analyses will advance our understanding of glycan biosynthesis and the evolution of breast milk.
PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexes at Nucleotide Resolution
Authors: Vihervaara Anniina , Versluis Philip , Lis John
Published: February 2023

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we developed Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq) and tracked phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncovered precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide, through early and late promoter-proximal pause, and into productive elongation. Pol II CTD was predominantly unphosphorylated in the early pause-region, whereas serine-2- and serine-5-phosphorylations occurred preferentially in the later pause-region. Serine-7-phosphorylation dominated after the pause-release in a region where Pol II accelerates to its full elongational speed. Interestingly, tracking transcription upon heat-induced reprogramming demonstrated that Pol II with phosphorylated CTD remains paused on heat-repressed genes.
NaV1.7 mRNA and protein expression in putative projection neurons of the human spinal dorsal horn
Authors: Shiers Stephanie , Funk Geoffrey , Cervantes Anna , Horton Peter , Dussor Gregory , Hennen Stephanie , Price Theodore J.
Published: February 2023

NaV1.7, a membrane-bound voltage-gated sodium channel, is preferentially expressed along primary sensory neurons, including their peripheral & central nerve endings, axons, and soma within the dorsal root ganglia and plays an integral role in amplifying membrane depolarization and pain neurotransmission. Loss- and gain-of-function mutations in the gene encoding NaV1.7,SCN9A, are associated with a complete loss of pain sensation or exacerbated pain in humans, respectively. As an enticing pain target supported by human genetic validation, many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials. The underlying reasons are still unclear, but recent reports suggest that inhibiting NaV1.7 in central terminals of nociceptor afferents is critical for achieving pain relief by pharmacological inhibition of NaV1.7. We report for the first time that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+) in the human spinal dorsal horn, predominantly in lamina 1 and 2, as well as in deep dorsal horn neurons and motor neurons in the ventral horn. NaV1.7 protein was found in the central axons of sensory neurons terminating in lamina 1-2, but also was detected in the axon initial segment of resident spinal dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with geneticSCN9Amutations, and in achieving analgesic efficacy in clinical trials.
Effect ofLantana camaraethanolic leaf extract on survival and migration of MDA-MB-231 triple negative breast cancer cell line
Authors: Pal Arundhaty , Sanyal Sourav , Das Sayantani , Sengupta Tapas Kumar
Published: February 2023

Introduction

Breast cancer is a leading cause of cancer-related death worldwide.Lantana camarahas been reported to cure a number of ailments, with few studies showing its cytotoxic effects on breast cancer cells. However, the impact ofLantana camaraon triple negative breast cancer cells is largely obscure to date. The present study investigated the effect of ethanolic extract ofLantana camaraleaves on the triple negative breast cancer cell line, MDA-MB-231.

Methods

Cytotoxic effect of the extract on the cells was determined by cell survival assay. Cell cycle phase distribution was analysed using flow cytometry. To study the effect on nuclear morphology, nuclear stained cells were visualized using epifluorescence microscopy. The induction of apoptosis and generation of reactive oxygen species was conducted using flow cytometry. Cellular migration was studied by performing wound healing assay. Real-time polymerase chain reaction was done to determine the mRNA levels of some key genes.

Results

Lantana camaraleaf extract induced cytomorphological changes and growth inhibitory effect on MDA-MB-231 cells in a dose-dependent manner. The extract also induced G0/G1 cell cycle arrest and nuclear condensation. Flow cytometry analysis confirmed cell death by apoptosis.Lantana camaraleaf extract also reduced the migration of MDA-MB-231 cells. mRNA expression levels also supported the above observations.

Conclusions

This study demonstrates the efficacy of the extract to induce growth-inhibitory and anti-migratory effects on MDA-MB-231 cells. Our results thus suggest thatLantana camaraleaf extract can be considered as a potent source of chemotherapeutic agents for triple negative breast cancer.
Transcriptional networks predating cognition-associated pyramidal lineages are restructured by erythropoietin
Authors: Singh Manvendra , Zhao Ying , Gastaldi Vinicius Daguano , Wojcik Sonja M. , Curto Yasmina , Kawaguchi Riki , Merino Ricardo M. , Garcia-Agudo Laura Fernandez , Taschenberger Holger , Brose Nils , Geschwind Daniel , Nave Klaus-Armin , Ehrenreich Hannelore
Published: February 2023

Recombinant human erythropoietin (rhEPO) has potent procognitive effects, hematopoiesis-independent, but underlying mechanisms and physiological role of brain-expressed EPO have remained obscure. Here, we provide encyclopedic transcriptional hippocampal profiling of mice treated with rhEPO. Based on ∼108,000 single nuclei, we unmask multiple pyramidal lineages with their comprehensive molecular signatures. By temporal profiling and gene regulatory analysis, we build a developmental trajectory of CA1 pyramidal neurons derived from multiple predecessor lineages and elucidate gene regulatory networks underlying their fate determination. With EPO as ꞌtoolꞌ, we discover novel populations of newly differentiating pyramidal neurons, overpopulating to ∼200% upon rhEPO with upregulation of genes crucial for neurodifferentiation, dendrite growth, synaptogenesis, memory formation, and cognition. Using a Cre-based approach to visually distinguish pre-existing from newly formed pyramidal neurons for patch-clamp recordings, we learn that rhEPO treatment differentially affects excitatory and inhibitory inputs. Our findings provide mechanistic insight into how EPO modulates neuronal functions and networks.
Conserved use of the sodium/bile acid cotransporter (NTCP) as an entry receptor by hepatitis B virus and domestic cat hepadnavirus
Authors: Shofa Maya , Ohkawa Akiho , Kaneko Yasuyuki , Saito Akatsuki
Published: February 2023

TheOrthohepadnavirusgenus includes hepatitis B virus (HBV) that can cause chronic hepatitis and hepatocarcinoma in humans. Recently, a novel hepadnavirus in cats, domestic cat hepadnavirus (DCH), was identified that is genetically close to HBV. DCH infection is associated with chronic hepatitis in cats, suggesting a similarity with HBV pathogenesis and the potential to use DCH as a novel animal model for HBV research. HBV is shown to use the sodium/bile acid cotransporter (NTCP) as a major cell entry receptor, but the equivalent receptor for DCH remains unknown. Here we sought to identify the entry receptor for DCH. HBV- and DCH-derived preS1 peptides efficiently bound to both human and cat NTCPs, and residue 158 of NTCP proteins determined the species-specific binding of the DCH preS1 peptide. Myrcludex B, an HBV entry inhibitor, blocked binding of the DCH preS1 peptide. Thus, DCH and HBV may share cell entry molecules, suggesting a possibility of inter-species transmission. Furthermore, our study suggests that DHC can be useful as a novel model for HBV research.
Microplastics analytics: why we should not underestimate the importance of blank controls
Authors: Noonan Michael J. , Ribeiro Nicole G. , Mills C Lauren , de A. M. M. Ferraz Marcia
Published: February 2023

Recent years have seen considerable scientific attention devoted towards documenting the presence of microplastics (MPs) in environmental samples. Due to omnipresence of environmental microplastics, however, disentangling environmental MPs from sample contamination is a challenge. Hence, the environmental (collection site and laboratory) microplastics contamination of samples during processing is a reality that we must address, in order to generate reproducible and reliable data. Here we investigated published literature and have found that around 1/5 of studies failed to use blank controls in their experiments. Additionally, only 34% of the studies used a controlled air environment for their samples processing (laminar flow, fume hood, closed laboratory, clean room, etc.). In that regard, we have also shown that preparing samples in the fume hood, leads to more microplastics contamination than preparing it in the laboratory bench and the laminar flow. Although it did not completely prevent microplastics contamination, the processing of sample inside the laminar flow is the best option to reduce sample contamination during processing. Overall, we showed that blank controls are a must in microplastics sample preparation, but it is often overlooked by researchers.

Highlights

Most of the contaminant microplastics in blank controls were particles < 20 μm.

Fume hoods result in more contamination than processing the samples on the bench.

Laminar flow was the best option for reducing MPs contamination of samples.

1/5 of studies failed to use blank controls, and 1/3 did not correct their data.

Improving the use and description of blanks is imperative for ensuring data quality.

Mandatory graphical abstract