title: Ligand Binding Prediction using Protein Structure Graphs and Residual Graph Attention Networks

abstract:
MotivationComputational prediction of ligand-target interactions is a crucial part of modern drug discovery as it helps to bypass high costs and labor demands of in vitro and in vivo screening. As the wealth of bioactivity data accumulates, it provides opportunities for the development of deep learning (DL) models with increasing predictive powers. Conventionally, such models were either limited to the use of very simplified representations of proteins or ineffective voxelization of their 3D structures. Herein, we present the development of the PSG-BAR (Protein Structure Graph -Binding Affinity Regression) approach that utilizes 3D structural information of the proteins along with 2D graph representations of ligands. The method also introduces attention scores to selectively weight protein regions that are most important for ligand binding.

ResultsThe developed approach demonstrates the state-of-the-art performance on several binding affinity benchmarking datasets. The attention-based pooling of protein graphs enables identification of surface residues as critical residues for protein-ligand binding. Finally, we validate our model predictions against an experimental assay on a viral main protease (Mpro)- the hallmark target of SARS-CoV-2 coronavirus.

AvailabilityThe code for PSG-BAR is made available at https://github.com/diamondspark/PSG-BAR

Contactacherkasov@prostatecentre.com


title: Stardust: improving spatial transcriptomics data analysis through space aware modularity optimization based clustering.

abstract:
BackgroundSpatial transcriptomics (ST) combines stained tissue images with spatially resolved high-throughput RNA sequencing. The spatial transcriptomic analysis includes challenging tasks like clustering, where a partition among data points (spots) is defined by means of a similarity measure. Improving clustering results is a key factor as clustering affects subsequent downstream analysis. State-of-the-art approaches group data by taking into account transcriptional similarity and some by exploiting spatial information as well. However, it is not yet clear how much the spatial information combined with transcriptomics improves the clustering result.

ResultsWe propose a new clustering method, Stardust, that easily exploits the combination of space and transcriptomic information in the clustering procedure through a manual or fully automatic tuning of algorithm parameters. Moreover, a parameter-free version of the method is also provided where the spatial contribution depends dynamically on the expression distances distribution in the space. We evaluated the proposed methods results by analysing ST datasets available on the 10x Genomics website and comparing clustering performances with state-of-the-art approaches by measuring the spots stability in the clusters and their biological coherence. Stability is defined by the tendency of each point to remain clustered with the same neighbours when perturbations are applied.

ConclusionsStardust is an easy-to-use methodology allowing to define how much spatial information should influence clustering on different tissues and achieving more stable results than state-of-the-art approaches.

category: bioinformatics


title: Impact of freeze-thaw-induced pit aspiration on stem water transport in a subalpine conifer (Abies veitchii)

abstract: During winter, subalpine conifers experience frequent freeze-thaw cycles in stem xylem, which may cause embolism and pit aspiration due to increased water volume during the sap to ice transition. This study examined the occurrence and ecological impacts of a combination of freeze-thaw-induced pit aspiration and embolism. In subalpine Abies veitchii trees, the fraction of closed pits and embolized tracheids as well as conductivity losses were measured to examine pit aspiration and its effects, triggered by natural and artificial stem freezing. When trees incurred mild drought stress in February and early March, 70% to 80% of stem conductivity was lost. Cryo-scanning electron microscopy indicated <20% embolized tracheids but [~]90% closed pits. Severe drought stress in late March caused 96 {+/-} 1.2% (mean {+/-} SE) loss of stem conductivity, while the fraction of embolized tracheids increased to 64 {+/-} 6.6%, and aspirated pit fraction decreased to 23 {+/-} 5.6%. Experimental freeze-thaw cycles also induced from 7.1 {+/-} 0.89% to 49 {+/-} 10% pit aspiration, and the fraction of closed pits was positively correlated to the percent loss of stem hydraulic conductivity. Results indicated that freezing-induced pit aspiration is an important factor for stem xylem dysfunction under mild drought. Upon severe drought in winter, stem water transport is predominantly inhibited by xylem embolism.

category: plant biology


title: A Pan-Coronavirus Vaccine Candidate: Nine Amino Acid Substitutions in the ORF1ab Gene Attenuate 99% of 365 Unique Coronaviruses: A Comparative Effectiveness Research Study

abstract:
BackgroundThe COVID-19 pandemic has been a watershed event. Industry and governments have reacted, investing over US$105 billion in vaccine research.1 The  Holy Grail is a universal, pan-coronavirus, vaccine to protect humankind from future SARS-CoV-2 variants and the thousands of similar coronaviruses with pandemic potential.2 This paper proposes a new vaccine candidate that appears to attenuate the SARS-Cov-2 coronavirus variants to render it safe to use as a vaccine. Moreover, these results indicate it may be efficacious against 99% of 365 coronaviruses. This research model is wet-dry-wet; it originated in genomic sequencing laboratories, evolved to computational modeling, and the candidate result now require validation back in a wet lab.

ObjectivesThis studys purpose was to test the hypothesis that machine learning applied to sequenced coronaviruses genomes could identify which amino acid substitutions likely attenuate the viruses to produce a safe and effective pan-coronavirus vaccine candidate. This candidate is now eligible to be pre-clinically then clinically tested and proven. If validated, it would constitute a traditional attenuated virus vaccine to protect against hundreds of coronaviruses, including the many future variants of SARS-CoV-2 predicted from continuously recombining in unvaccinated populations and spreading by modern mass travel.

MethodsUsing machine learning, this was an in silico comparative effectiveness research study on trinucleotide functions in nonstructural proteins of 365 novel coronavirus genomes. Sequences of 7,097 codons in the ORF1ab gene were collected from 65 global locations infecting 68 species and reported to the US National Institute of Health. The data were proprietarily transformed twice to enable machine learning ingestion, mapping, and interpretation. The set of 2,590,405 data points was randomly divided into three cohorts: 255 (70%) observations for training; and two cohorts of 55 (15%) observations each for testing. Machine learning models were trained in the statistical programming language R and compared to identify which mixture of the 7.097 x 1023 possible amino-acid-location combinations would attenuate SARS-CoV-2 and other coronaviruses that have infected humans.

ResultsContests of machine-learning algorithms identified nine amino-acid point substitutions in the ORF1ab gene that likely attenuate 98.98% of 365 (361) novel coronaviruses. Notably, seven substitutions are for the amino acid alanine. Most of the locations (5 of 9) are in nonstructural proteins (NSPs) 2 and 3. The substitutions are alanine to (1) valine at codon 4273; (2) leucine at codon 5077; (3) phenylalanine at codon 2001; (4) leucine at codon 372; (5) proline at codon 354; (6) phenylalanine at codon 2811; (7) phenylalanine at codon 4703; (8) leucine to serine at codon 2333; and, (9) threonine to alanine at codon 5131.

ConclusionsThe primary outcome is a new, highly promising, pan-coronavirus vaccine candidate based on nine amino-acid substitutions in the ORF1ab gene. The secondary outcome was evidence that sequences of wet-dry lab collaborations - here machine learning analysis of viral genomes informing codon functions -- may discover new broader and more stable vaccines candidates more quickly and inexpensively than traditional methods.

category: synthetic biology


title: Two types of human TCR differentially regulate reactivity to self and non-self antigens

abstract: Based on analyses of TCR sequences from over 1,000 individuals, we report that the TCR repertoire is composed of two ontogenically and functionally distinct types of TCRs. Their production is regulated by variations in thymic output and terminal deoxynucleotidyl transferase (TDT) activity. Neonatal TCRs derived from TDT-negative progenitors persist throughout life, are highly shared among subjects, and are polyreactive to self and microbial antigens. Thus, >50% of cord blood TCRs are responsive to SARS-CoV2 and other common pathogens. TDT- dependent TCRs present distinct structural features and are less shared among subjects. TDT- dependent TCRs are produced in maximal numbers during infancy when thymic output and TDT activity reach a summit, are more abundant in subjects with AIRE mutations, and seem to play a dominant role in graft-versus-host disease. Factors decreasing thymic output (age, male sex) negatively impact TCR diversity. Males compensate for their lower repertoire diversity via hyperexpansion of selected TCR clonotypes.

category: immunology


title: GM-CSF-activated human dendritic cells promote type1 T follicular helper cells (Tfh1) polarization in a CD40-dependent manner

abstract:
T follicular helper (Tfh) cells are specialized CD4+ T cells that regulate humoral immunity by providing B cell help. Tfh1 sub-population was recently identified and associated with severity in infection and autoimmune diseases. The cellular and molecular requirements to induce human Tfh1 differentiation are unknown. Our work investigated the role of human dendritic cells (DC) in promoting Tfh1 differentiation and their physiopathological implication in mycobacterium tuberculosis and mild COVID-19 infection.

Activated human blood CD1c+ DC were cocultured with allogeneic naive CD4+ T cells. Single-cell RNA sequencing was then used alongside protein validation to define the induced Tfh lineage. DC signature and correlation with Tfh1 cells in infected patients was established through bioinformatic analysis.

Our results show that GM-CSF-activated DC drove the differentiation of Tfh1 cells, displaying typical Tfh molecular features, including 1) high levels of PD-1, CXCR5, and ICOS expression; 2) BCL6 and TBET co-expression; 3) IL-21 and IFN-{gamma} secretion. Mechanistically, GM-CSF triggered the emergence of two distinct DC sub-populations defined by their differential expression of CD40 and ICOS-ligand (ICOS-L), and distinct phenotype, morphology, transcriptomic signature, and function. We showed that Tfh1 differentiation was efficiently and specifically induced by CD40highICOS-Llow DC in a CD40-dependent manner. Tfh1 cells were positively associated with a CD40highICOS-LLow DC signature in patients with latent mycobacterium tuberculosis and mild COVID-19 infection.

Our study uncovers a novel CD40-dependent human Tfh1 axis. Immunotherapy modulation of Tfh1 activity might contribute to control diseases where Tfh1 are known to play a key role, such as infections.

Significance StatementDendritic cells (DC) play a central role in triggering the adaptive immune response due to their T cell priming functions. Among different T cell subsets, it is still not clear how human type1 T follicular helper cells (Tfh1) differentiate. Tfh1 cells are implicated in several physiopathological conditions, including infections. Here we show that GM-CSF induces diversification of human DC. Only CD40highICOS-LLow DC were able to drive Tfh1 cell differentiation. We found that CD40highICOS-LLow DC signature was associated to Tfh1 cells in mycobacterium tuberculosis and COVID-19 patients. Our data reveal a previously undescribed pathway leading to human Tfh1 cell differentiation and highlight the importance of GM-CSF and CD40 as potential targets for the design of anti-infective therapies.


title: Structural and functional characteristics of SARS-CoV-2 Omicron subvariant BA.2 spike

abstract: The Omicron subvariant BA.2 has become the dominant circulating strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in many countries. We have characterized structural, functional and antigenic properties of the full-length BA.2 spike (S) protein and compared replication of the authentic virus in cell culture and animal model with previously prevalent variants. BA.2 S can fuse membranes more efficiently than Omicron BA.1, mainly due to lack of a BA.1-specific mutation that may retard the receptor engagement, but still less efficiently than other variants. Both BA.1 and BA.2 viruses replicated substantially faster in animal lungs than the early G614 (B.1) strain in the absence of pre-existing immunity, possibly explaining the increased transmissibility despite their functionally compromised spikes. As in BA.1, mutations in the BA.2 S remodel its antigenic surfaces leading to strong resistance to neutralizing antibodies. These results suggest that both immune evasion and replicative advantage may contribute to the heightened transmissibility for the Omicron subvariants.

category: microbiology


title: The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype

abstract: Variant of concern (VOC) Omicron-BA1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and multiple animal models is urgently needed. Here, we characterized Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in naive hamsters, ferrets and hACE2-expressing mice, and in immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In Syrian hamsters, Delta showed dominance over Omicron-BA.1 and in ferrets, Omicron-BA.1 infection was abortive. In mice expressing the authentic hACE2-receptor, Delta and a Delta spike clone also showed dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naive K18-hACE2 mice, we observed Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of both Delta and Omicron-BA.1 replication and pathogenicity. Finally, the Omicron-BA.1 spike clone was less well controlled by mRNA-vaccination in K18-hACE2-mice and became more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance.


title: Chromoanagenesis in the asy1 meiotic mutant of Arabidopsis

abstract: Chromoanagenesis is a catastrophic event that involves localized chromosomal shattering and reorganization. In this study, we report a case of chromoanagenesis resulting from defective meiosis in the MEIOTIC ASYNAPTIC MUTANT 1 (asy1) background in Arabidopsis thaliana. We provide a detailed characterization of the genomic structure of this individual with a severely shattered segment of chromosome 1. We reveal more than 300 candidate novel DNA junctions in the affected region, confirming that asy1-related defective meiosis is a potential trigger for chromoanagenesis. This is the first example of chromoanagenesis associated with female meiosis and indicates the potential for genome evolution during oogenesis.

category: genomics


title: Characterization of the cluster MabR prophages of Mycobacterium abscessus and Mycobacterium chelonae

abstract: Mycobacterium abscessus is an emerging pathogen of concern in cystic fibrosis and immunocompromised patients and is considered one of the most drug-resistant mycobacteria. The majority of clinical M. abscessus isolates carry one or more prophages that are hypothesized to contribute to virulence and bacterial fitness. The prophage McProf was identified in the genome of the Bergey strain of M. chelonae, and is distinct from previously described prophages of M. abscessus. The McProf genome increases intrinsic antibiotic resistance of M. chelonae and drives expression of the intrinsic antibiotic resistance gene, whiB7, when superinfected by a second phage. The prevalence of McProf-like genomes was determined in sequenced mycobacterial genomes. Related prophage genomes were identified in the genomes of 25 clinical isolates of M. abscessus and assigned to the novel cluster, MabR. The MabR genomes share less than 10% gene content with previously described prophages; however, share features typical of prophages, including polymorphic toxin immunity (PT-Imm) systems.


title: Hyperactive somatostatin interneurons near amyloid plaque and cell-type-specific firing deficits in a mouse model of Alzheimer's disease

abstract: Alzheimers disease (AD) is characterized by synaptic loss and neuronal network dysfunction. These network deficits are mediated by early alterations in neuronal firing rates that coincide with amyloid plaque accumulation. Mounting evidence supports that inhibitory networks are impaired in AD, but the mechanisms driving these inhibitory deficits are poorly understood. Here we use in vivo multiphoton calcium imaging to determine the relationship between amyloid accumulation and the spontaneous activity of excitatory neurons and inhibitory interneurons in an APP/PS1 mouse model of Alzheimers disease. We show that somatostatin-expressing (SOM) interneurons are hyperactive, while parvalbumin-expressing interneurons are hypoactive in APP/PS1 mice. Only SOM interneuron hyperactivity correlated with proximity to amyloid plaque. These inhibitory deficits were accompanied by decreased excitatory neurons activity and decreased pairwise activity correlations in APP/PS1 mice. Our study identifies cell-specific interneuronal firing deficits driven by amyloid pathology in APP/PS1 mice and provides new insights for targeting inhibitory circuits in Alzheimers disease.

category: neuroscience


title: A Neuropeptidergic Signaling Pathway for Olfactory Gain Modulation

abstract: Sensory systems are dynamically adjusted according to the animals ongoing needs by neuromodulators, such as neuropeptides. Despite their prevalence across all nervous systems1,2, how peptidergic neurons and the neuropeptide they release act to adjust sensory processing remains poorly understood. Here, we reveal that a heterogeneous ensemble of local interneurons (LNs) release the neuropeptide myoinhibitory peptide (MIP) within the Drosophila primary olfactory center (the antennal lobe, AL). We find MIPergic LNs form reciprocal connections with many AL principal neurons, but only a subset express the inhibitory MIP receptor (sex peptide receptor, SPR). We find both the MIPergic LNs and several of the SPR-expressing neurons are activated by the food-related odor apple cider vinegar (ACV), therefore suggesting MIP plays a role in adjusting ACV responses within the AL. We demonstrate that MIP can simultaneously decrease olfactory input to some glomeruli, while indirectly increasing olfactory input to others. As those glomeruli boosted by MIP - as well as MIP itself - are necessary and sufficient to initiate an attractive behavioral response to ACV3,4, the neural substrates identified here may represent a key circuit element for the animals switch in behavioral responses.


title: Persistence and in vivo evolution of vaginal bacterial strains over a multi-year time period

abstract:
It is not clear if the bacterial strains which comprise our microbiota are mostly long-term colonizers or transient residents. Studies have demonstrated decades long persistence of bacterial strains within the gut, but persistence at other body sites has yet to be determined. The vaginal microbiota (VMB) is often dominated by Lactobacillus, although it is also commonly comprised of a more diverse set of other facultative and obligate anaerobes. Longitudinal studies have demonstrated that these communities can be stable over several menstrual cycles or can fluctuate temporally in species composition. We sought to determine whether the bacterial strains which comprise the VMB were capable of persisting over longer time-periods. We performed shotgun metagenomics on paired samples from 10 participants collected 1 and 2 years apart. The resulting sequences were de novo assembled and binned into high-quality metagenome assembled genomes. Persistent strains were identified based on the sequence similarity between the genomes present at the two timepoints and were found in the VMB of six of the participants, three of which had multiple. The VMB of the remaining four participants was similar in species composition at the two timepoints but was comprised of different strains. For the persistent strains, we were able to identify the mutations which fixed in the populations over the observed time period, giving insight into the evolution of these bacteria. These results indicate that bacterial strains can persist in the vagina for extended periods of time, providing an opportunity for them to evolve in the host microenvironment.

ImportanceThe persistence of strains within the vaginal microbiota has not yet been characterized. Should these strains be capable of persisting for extended periods of time, they could evolve within their host in response to selective pressures exerted by the host or by other members of the community. Here, we present preliminary findings which demonstrate that bacterial strains can persist in the vagina for at least one year and evolve over time. In several cases, multiple strains persisted together in a community, indicating that co-evolution between bacterial strains could occur in the vagina. Our observations motivate future studies which collect samples from more participants, at more timepoints and over even longer periods of time. Understanding which strains persist, what factors drive their persistence, and what selective pressures they face will inform the development and delivery of rationally designed live biotherapeutics for the vagina.


title: An essential host dietary fatty acid promotes TcpH inhibition of TcpP proteolysis enabling virulence gene expression in Vibrio cholerae

abstract:
Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated indirectly by two single-pass membrane-localized transcription regulators (MLTR), ToxR and TcpP, that promote expression of the transcription activator toxT. TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. Absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that a dietary fatty acid, -linolenic acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs; also known as lipid rafts) in a mechanism requiring the TcpH transmembrane domain. Taken together our data support a model where V. cholerae cells use exogenous -linolenic acid to remodel the phospholipid bilayer in vivo, leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity.

Significance StatementV. cholerae continues to pose a significant global burden on health infection millions of people every year resulting in [~]100,000 deaths annually. The importance of toxT gene expression in V. cholerae pathogenesis has been well established. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction, in the presence of -linolenic acid, an essential dietary fatty acid. Here we identify a physiological relevant host factor that stimulates V. cholerae pathogenicity via TcpH-dependent antagonism of TcpP proteolysis.


title: IgaA protein, GumB, has a global impact on the transcriptome and surface proteome of Serratia marcescens

abstract:
Bacterial stress response signaling systems, like the Rcs system, can be triggered by membrane and cell wall damaging compounds including antibiotics and innate immune system factors. These regulatory systems help bacteria survive envelope stress by altering the transcriptome resulting in protective phenotypic changes that may also the influence the virulence of the bacterium. This study investigated the role of the Rcs stress response system using a clinical keratitis isolate of S. marcescens with a mutation in the gumB gene. GumB, an IgaA ortholog, inhibits activation of the Rcs system, such that mutants have overactive Rcs signaling. Transcriptomic analysis indicated that approximately 15% of all S. marcescens genes were significantly altered with two-fold or greater changes in expression in the {Delta}gumB mutant compared to the wild type indicating a global transcriptional regulatory role for GumB. We further investigated the phenotypic consequences of two classes of genes with altered expression in the {Delta}gumB mutant expected to contribute to infections: serralysin metalloproteases PrtS, SlpB and SlpE, and type I pili coded by fimABCD. Secreted fractions from the {Delta}gumB mutant had reduced cytotoxicity to a corneal cell line, and could be complemented by induced expression of prtS, but not cytolysin shlBA, phospholipase phlAB, or flagellar master regulator flhDC operons. Proteomic analysis, qRT-PCR, and type I pili dependent yeast agglutination indicated an inhibitory role for the Rcs system in adhesin production. Together these data demonstrate that GumB and the Rcs stress response system control S. marcescens virulence factors beyond the ShlA cytolysin.

IMPORTANCEPrevious studies indicate that the bacterial Rcs system is a key regulator of envelope stress. This study demonstrated that activation of the Rcs system had a global impact on the transcriptome of a clinical isolate of S. marcescens including decreased expression of cytotoxic serralysin metalloproteases and biofilm promoting type I pili. These results give mechanistic insight into how the Rcs system contributes to pathogenesis.


title: Tackling Recalcitrant Pseudomonas aeruginosa Infections In Critical Illness via Anti-virulence Monotherapy

abstract: Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report novel potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs) resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This novel class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.


title: Different Routes for Direct Interspecies Electron Transfer with Diverse Electron-Accepting Partners

abstract: Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but studies to date that have examined the mechanisms for electron transfer from electron-donating partners have primarily focused on co-cultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET-based growth with Geobacter sulfurreducens reducing fumarate was compared with the transcriptome of G. metallireducens grown in co-culture with diverse Methanosarcina species. The transcriptome of G. metallireducens co-cultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer-surface cytochromes, was different from the transcriptome of G. metallireducens co-cultured with Methanosarcina acetivorans or Methanosarcina subterranea, which have an outer-surface c-type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for c-type cytochrome genes were particularly notable. The impact of deleting G. metallireducens c-type cytochrome genes depended upon the electron-accepting partner. Multiple cytochromes that G. metallireducens required for DIET with G. sulfurreducens and for Fe(III) oxide reduction were not required for DIET with the Methanosarcina species. Porin-cytochrome complexes and e-pili were required for optimal DIET with all partners. Shewanella oneidensis, another electroactive microbe with abundant outer-surface c-type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer-surface cytochromes does not necessarily confer the capacity for DIET, and also emphasize the substantial impact of the physiology of the electron-accepting partner on the physiology of the electron-donating DIET partner.


title: Conserved FimK truncation coincides with increased expression of type 3 fimbriae and cultured bladder epithelial cell association in Klebsiella quasipneumoniae

abstract:
Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its urovirulence mechanisms have not been defined. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well-conserved between Escherichia coli and K. pneumoniae with the exception of fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that the bladder epithelial cell association and invasion of uropathogenic K. quasipneumoniae strain KqPF9 is dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52 and that complementation of KqPF9{Delta}fimK with the TOP52 fimK allele reduced type 3 fimbrial expression and bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation.

ImportanceK. quasipneumoniae is often indicated as the cause of many opportunistic infections including urinary tract infection (UTI), which affects >50% of women worldwide. However, virulence mechanisms of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae virulence phenotypes by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment. We identify clear phenotypic differences in both bladder epithelial cell attachment between uropathogenic K. pneumoniae and K. quasipneumoniae. Importantly, we find that a structural difference in the fimbrial regulatory gene fimK may contribute to differential co-regulation of type 1 and type 3 fimbriae between the two Klebsiella species.


title: Prolonged exposure to insulin causes epigenetic alteration leading to insulin resistance

abstract:
Glucose homeostasis is maintained by insulin. It has been observed that hyperinsulinemia precedes insulin resistance and Type 2 diabetes. Insulin resistance is caused by multiple factors including genetic and diet. The molecular mechanism underlying insulin resistance (IR) is not completely understood. Using Glut4 and insulin receptor-expressing CHO cells we had previously shown that prolonged exposure of these cells to insulin in the absence of high levels of glucose led to insulin resistance in the cells. In the present study, we have shown that the underlying cause for the impaired GLUT4 trafficking is the defective PI3K/AKT pathway. This insulin resistance is likely due to epigenetic alterations as it is stable and can be maintained for several generations even when insulin is not provided, and epigenetic modifiers can reverse the insulin resistance. We extended these studies to liver cell line (BRL-3A) and show that these cells also develop impaired insulin signaling upon exposure to insulin in the absence of high levels of glucose. Transcriptomic analysis of the insulin-sensitive and -resistance cells uncover altered signaling networks involved in chromatin remodelling, Rho GTPases, and ubiquitination. Pathway analysis reveals the role of demethylase Kdm5b and lysine methyltransferase (Kmt2a and Kmt2e) in the development of insulin resistance. It is also observed that trimethylation of histone H3 at lysine 4 (H3K4me3) is increased in insulin resistance cellular models. We further showed that mice injected with low doses of insulin when fasting develop insulin resistance with impaired glucose tolerance and increased HOMA-IR index. Altogether, these findings suggest dysregulated synthesis of insulin in the absence of glucose stimulus could lead to epigenetic alterations that may lead to insulin resistance.

Summary StatementInsulin stimulation in the absence of glucose leads to insulin resistance. We have developed a cell and mouse model of insulin resistance in this study to characterise the molecular signalling involved in insulin resistance and early onset of type 2 diabetes. The transcriptomic analysis provides new insights on epi-transcriptomic regulation in insulin resistance.

category: molecular biology


title: MTALTND4, a second protein coded by nd4 impacts mitochondrial bioenergetics

abstract: Recent evidence suggests that the coding potential of the mitogenome is underestimated. We found a downstream alternative ATG initiation codon in the +3 reading frame of the human mitochondrial nd4 gene. This newly characterized alternative open reading frame (altORF) encodes a 99-amino acids long polypeptide, MTALTND4, which is conserved in primates. This small protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts mitochondrial physiology. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.


title: The antibiotic phazolicin displays a dual mode of uptake in Gram-negative bacteria

abstract:
Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides.

SIGNIFICANCEMany bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti. This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.


title: The 2',3' cyclic phosphatase Angel1 facilitates mRNA degradation during human ribosome-associated quality control

abstract: The Ribosome-associated Quality Control (RQC) pathway serves to resolve ribosomes stalled during the translation process and degrade the associated mRNA and nascent polypeptide. Here we identify the 2,3 cyclic phosphatase Angel1 as a rate-limiting factor for this process in human cells. Angel1 associates with proteins of the RQC pathway and with mRNA coding regions, consistent with a factor that monitors the translation process. Depletion of Angel1 causes stabilization of reporter mRNAs that are targeted for RQC by the absence of stop codons, but not an mRNA targeted for nonsense-mediated decay. Angel1 catalytic activity is critical for its function in RQC, as a catalytic inactivating mutation causes loss of RQC function. We also identify N4BP2 as the human RQC endonuclease. Given the biochemical activity of Angel1 as a 2,3 cyclic phosphatase, our findings suggest that a rate-limiting step in RQC-mediated mRNA decay is the resolution of a cyclic phosphate, possibly one generated upon N4BP2 cleavage.


title: A human embryonic limb cell atlas resolved in space and time

abstract: Human limbs emerge during the fourth post-conception week as mesenchymal buds which develop into fully-formed limbs over the subsequent months. Limb development is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common. Decades of work with model organisms has outlined the fundamental processes underlying vertebrate limb development, but an in-depth characterisation of this process in humans has yet to be performed. Here we detail the development of the human embryonic limb across space and time, using both single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells, progressing from a restricted number of multipotent progenitors to myriad mature cell states, and identify several novel cell populations, including perineural fibroblasts and multiple distinct mesenchymal states. We uncover two waves of human muscle development, each characterised by different cell states regulated by separate gene expression programmes. We identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity and validate this by performing MSC knock down in human embryonic myoblasts, which results in significant upregulation of late myogenic genes. Spatially mapping the cell types of the limb across a range of gestational ages demonstrates a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncovers two transcriptionally and spatially distinct populations of the progress zone, which we term "outer" and "transitional" layers. The latter exhibits a transcriptomic profile similar to that of the chondrocyte lineage, but lacking the key chondrogenic transcription factors SOX5,6 & 9. Finally, we perform scRNA-seq on murine embryonic limbs to facilitate cross-species developmental comparison at single-cell resolution, finding substantial homology between the two species.

category: developmental biology


title: Candidate Genes Underlying Wolbachia-Associated Plastic Recombination Revealed by Ovarian Transcriptomics of D. melanogaster

abstract:
Phenotypic plasticity is prevalent in nature and can facilitate the acclimation of organisms to changing environments. Recombination rate is plastic in a diversity of organisms and under a variety of stressful conditions. However, the recent finding that Wolbachia pipientis induces plastic recombination in Drosophila melanogaster was surprising because Wolbachia is not strictly considered a stressor to this host. We investigate the molecular mechanisms of Wolbachia-associated plastic recombination by comparing the ovarian transcriptomes of D. melanogaster infected and uninfected with Wolbachia. Our data suggest that infection explains a small amount of transcriptional variation but specifically affects genes related to cell cycle, translation, and metabolism. We also find enrichment of cell division and recombination processes among genes with infection-associated differential expression. Broadly, the transcriptomic changes identified in this study provide insight for the mechanisms of microbe-mediated plastic recombination, an important but poorly understood facet of host-microbe dynamics.

Significance StatementThough it is documented that Wolbachia is associated with increased recombination in D. melanogaster, the underlying mechanisms remain unknown. Here we use ovarian transcriptomics in Wolbachia-infected and uninfected flies to identify candidate genes underlying Wolbachia-associated plastic recombination. We find that infection alters ovarian gene expression in subtle ways, and also identify exciting candidate genes for functional analysis in subsequent work. These candidate genes may be the first step in determining the molecular mechanisms underlying Wolbachia-associated plastic recombination. Moreover, our data contribute to the growing body of knowledge surrounding how Wolbachia affects host gene expression, and highlights how context-dependent these effects are.

category: evolutionary biology


title: Phenotyping technology for assessing protein content in seaweed by field spectroscopy and a machine learning algorithm

abstract:
Determining seaweed protein concentration and the associated phenotype is critical for food industries that require precise tools to moderate concentration fluctuations and attenuate risks. Algal protein extraction and profiling have been widely investigated, but content determination involves a costly, time-consuming, and high-energy, laboratory-based fractionation technique. The present study examines the potential of field spectroscopy technology as a precise, high-throughput, non-destructive tool for on-site detection of red seaweed protein concentration. By using information from a large dataset of 144 Gracilaria sp. specimens, studied in a land-based cultivation set-up, under six treatment regimes during two cultivation seasons, and an artificial neural network, machine learning algorithm and diffuse visible-near infrared reflectance spectroscopy, predicted protein concentrations in the algae were obtained. The prediction results were highly accurate (R2 = 0.95; RMSE = 0.84), exhibiting a high correlation with the analytically determined values. External validation of the model derived from a separate trial, exhibited even better results (R2 = 0.99; RMSE = 0.45). This model, trained to convert phenotypic spectral measurements and pigment intensity into accurate protein content predictions, can be adapted to include diversified algae species and usages.

HighlightNon-destructive determination of protein content in the edible red seaweed Gracilaria sp. by in-situ, VIS-NIR spectroscopy and a machine learning algorithm.

category: biochemistry


title: An auto-inhibited state of protein kinase G and implications for selective activation

abstract: Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Campaigns targeting nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis suggest that understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG I and I{beta} that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is still not well understood. Here we present a crystal structure of PKG I{beta} in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG I{beta} auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG I{beta} auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and activation of PKGs.


title: Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

abstract: Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants and this is the most frequently applied process for the generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system localized in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2[A] resolution and that of the related plasmid pKM101-determined N-pilus at 3[A] resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens and TraM in pKM101) and phospholipids arranged in a 5-start helical assembly. They contain positively charged amino acids in the pilus lumen and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge to the lumen. Mutagenesis of the lumen-exposed Arg91 residue in VirB2 resulted in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion system pili and that the charge of the lumen is of functional importance.


title: Allosteric inhibition of PRPS is moderated by filamentous polymerization

abstract: Phosphoribosyl pyrophosphate (PRPP) is an important intermediate for the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) has been associated with human disorders including Arts syndrome, retinal dystrophy and gouty arthritis. Recent studies have revealed that PRPS can form filamentous cytoophidia in prokaryotes and eukaryotes. Here we resolve two distinct filament structures of E. coli PRPS at near-atomic resolution under Cryo-EM. Formation of two types of filaments is controlled by the binding of different ligands. While the type A filament attenuates the allosteric inhibition of PRPS by ADP, the type B filament enhances the inhibition. In addition, a novel conformation of the regulatory flexible loop of PRPS was found occupying the ATP binding site. AMP/ADP bound at a noncanonical allosteric site interacts with the regulatory flexible loop and facilitates the binding of ATP. Our findings not only reveal molecular mechanisms of the regulation of PRPS with structural basis, but also suggest a distinctive bidirectional regulatory system for PRPP production via PRPS polymerization.


title: The disordered N-terminus of McdB modulates phase separation via pH-sensitive hydration and folds upon interaction with McdA

abstract:
Carboxysomes are protein-based organelles essential for efficient CO2-fixation in cyanobacteria and some chemoautotrophic bacteria. We recently identified the two-component system responsible for spatially regulating carboxysomes, consisting of the proteins McdA and McdB. McdA is a member of the ParA/MinD-family of ATPases, which position a variety of cellular cargos across bacteria. McdB, however, represents a widespread but unstudied class of proteins. We previously found that McdB forms a hexamer and undergoes robust Liquid-Liquid Phase Separation (LLPS) in vitro, but the sequence and structural determinants underlying these properties are unknown. Here we define the domain architecture for McdB from the model cyanobacterium S. elongatus PCC 7942 which we use to dissect McdB oligomerization and LLPS. We identify an N-terminal Intrinsically Disordered Region (IDR), a central Q-rich dimerizing domain, and a C-terminal domain that trimerizes McdB dimers. Intriguingly, all three domains contributed to McdB LLPS. The Q-rich domain drove LLPS, the IDR tuned solubility, and the C-terminal domain provided further oligomerization to achieve full-length LLPS activity. We also identified critical basic residues in the IDR that modulate McdB LLPS, which we mutate to fine-tune condensate solubility both in vitro and in vivo. Our findings show that IDRs are not always drivers of LLPS, but can play secondary roles in modulating condensate solubility. Finally, we provide in silico evidence suggesting the N-terminal IDR of McdB acts as a MoRF, folding upon interaction with McdA. The data advance our understanding and application of carboxysomes, their positioning system, and the molecular grammar governing protein phase separation.

SIGNIFICANCEThe recently characterized Maintenance of Carboxysome Distribution (Mcd) system is responsible for spatially regulating carbon-fixing organelles in bacteria called carboxysomes. Although an understanding of the Mcd system would advance our application of carboxysomes to help engineer carbon-fixing organisms and combat the climate crisis, one of its two essential components, McdB, has only recently been identified and is poorly understood. Here, we provide a thorough biochemical characterization of McdB from the model cyanobacterium S. elognatus. We define a structural model for McdB and identify how specific domains and residues contribute to its oligomerization and phase separation. Notably, we saw that a disordered region of McdB regulates phase separation in response to pH; impactful to both carboxysome regulation and protein phase separation.


title: An Engineered Nanocomposite Copper Coating with Enhanced Antibacterial Efficacy

abstract:
Contaminated surfaces are a major source of nosocomial infection. To reduce microbial bioburden and surface-based transmission of infectious disease, the use of antibacterial and self-sanitizing surfaces, such as copper (Cu), is being explored in clinical settings. Cu has long been known to have antimicrobial activity. However, Gram-positive microorganisms, a class that includes pathogens commonly responsible for hospital-acquired infection such as Staphylococcus aureus and Clostridioides difficile, are more resilient to its biocidal effect. Inspired by inherently bactericidal nanostructured surfaces found in nature, we have developed an improved Cu coating, engineered to contain nanoscale surface features and thus increase its antibacterial activity against a broader range of organisms. In addition, we have established a new method for facilitating the rapid and continuous release of biocidal metal ions from the coating, through incorporation of an antibacterial metal salt (ZnCl2) with a lower reduction potential than Cu. Electrophoretic deposition (EPD) was used to fabricate our coatings, which serves as a low-cost and scalable route for modifying existing conductive surfaces with complex shape. By tuning both the surface morphology and chemistry, we were able to create a nanocomposite Cu coating that decreased the microbial bioburden of Gram-positive S.aureus by 94% compared to unmodified Cu.

Table of Contents O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Antimicrobial copper (Cu) products are being deployed in clinical settings to decrease microbial bioburden and prevent surface-based transmission of infectious disease. However, Gram-positive bacteria demonstrate increased resistance to Cus biocidal effects. To improve Cus antibacterial efficacy against Gram-positive bacteria, we have developed a hydrophobic Cu coating with cytotoxic nanotopography that facilitates the rapid and continuous release of biocidal metal ions.

C_FIG_DISPLAY

category: bioengineering


title: Quantitative analysis of surface wave patterns of Min proteins

abstract: The Min protein system is arguably the best-studied model systems for biological pattern formation. It exhibits pole-to-pole oscillations in E. coli bacteria as well as a variety of surface wave patterns in in vitro reconstitutions. Such Min surface wave patterns pose particular challenges to quantification as they are typically only semi-periodic and non-stationary. Here, we present a methodology for quantitatively analyzing such Min patterns, aiming for reproducibility, user-independence, and easy usage. After introducing pattern-feature definitions and image-processing concepts, we present an analysis pipeline where we use autocorrelation analysis to extract global parameters such as the average spatial wavelength and oscillation period. Subsequently, we describe a method that uses flow-field analysis to extract local properties such as the wave propagation velocity. We provide descriptions on how to practically implement these quantification tools and provide Python code that can directly be used to perform analysis of Min patterns.

category: biophysics


title: The "LINC" in between Δ40p53-miRNA axis in the regulation of cellular processes

abstract:
Recently, we showed that {Delta}40p53, the translational isoform of p53, can inhibit cell growth independently of p53 by regulating microRNAs. Here, we explored the role of {Delta}40p53 in regulating the lncRNA-miRNA axis. Based on early research from our laboratory, we selected LINC00176 for further exploration. LINC00176 levels were affected by the overexpression and knockdown of {Delta}40p53. Further, under DNA damage, ER stress, and glucose deprivation, LINC00176 was upregulated in HCT116 p53-/- cells (harboring only {Delta}40p53) compared to HCT116 p53+/+ cells. Additionally, ChIP and RNA stability assays revealed that {Delta}40p53 transactivates LINC00176 transcriptionally and stabilizes it post-transcriptionally. We ectopically overexpressed and knocked down LINC00176 in HCT116 p53-/- cells, which affected proliferation, cell viability, and the expression of epithelial markers. Finally, RNA pulldown revealed that LINC00176 sequesters several putative miRNA targets. These results provide important insights into the pivotal role of {Delta}40p53 in regulating the lncRNA-miRNA axis.

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=171 HEIGHT=200 SRC="FIGDIR/small/489891v2_ufig1.gif" ALT="Figure 1">
View larger version (31K):
org.highwire.dtl.DTLVardef@1035f85org.highwire.dtl.DTLVardef@74185borg.highwire.dtl.DTLVardef@1eb3704org.highwire.dtl.DTLVardef@168ebab_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Model depicting the regulation of {Delta}40p53-LINC00176-miRNA axis

(A) In absence of {Delta}40p53, LINC00176 levels do not increase which leads to increased pool of free miRNAs which probably binds to several mRNA targets preventing their function, ultimately resulting in increased cell proliferation. (B) However, when {Delta}40p53 is present it upregulates LINC00176 levels both at the level of transcription and post-transcription, which sequesters oncogenic miRNAs preventing it to bind to their respective mRNA targets, which ultimately results in decreased cell proliferation.

C_FIG

category: cancer biology


title: The "LINC" in between Delta40p53-miRNA axis in the regulation of cellular processes


title: Molecular Characterization of the Tumor Microenvironment in Renal Medullary Carcinoma

abstract: Renal medullary carcinoma (RMC) is a highly aggressive disease associated with sickle hemoglobinopathies and universal loss of the tumor suppressor gene SMARCB1. RMC has a relatively low rate of incidence compared with other renal cell carcinomas (RCCs) that has hitherto made molecular profiling difficult. To probe this rare disease in detail we performed an in-depth characterization of the RMC tumor microenvironment using a combination of genomic, metabolic and single-cell RNA-sequencing experiments on tissue from a representative untreated RMC patient, complemented by retrospective analyses of archival tissue and existing published data. Our study of the tumor identifies a heterogenous population of malignant cell states originating from the thick ascending limb of the Loop of Henle within the renal medulla, displaying the hallmarks of increased resistance to cell death by ferroptosis and proteotoxic stress driven by MYC-induced proliferative signals. Specifically, genomic characterization of RMC tumors provides substantiating evidence for the recently proposed dependence of SMARCB1-difficient cancers on an intact CDKN2A-p53 pathway and we suggest increased cystine-mTORC-GPX4 signaling also plays a role within transformed RMC cells. We further propose that RMC has an immune landscape comparable to that of untreated RCCs, including heterogenous expression of the immune ligand CD70 within a sub-population of tumor cells, which could provide an immune-modulatory role that serves as a viable candidate for therapeutic targeting.


title: ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression

abstract: The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that the ST6GAL1 sialyltransferase, which adds 2,6-linked sialic acids to N-glycosylated proteins, is upregulated in patients with early-stage PDAC, and further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft models with human PDAC cells. Additionally, we developed a genetically-engineered mouse (GEM) with transgenic expression of ST6GAL1 in the pancreas, and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D have greatly accelerated PDAC progression and mortality compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1s role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We confirmed that ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a novel glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.


title: RNA-seq sample preparation kits strongly affect transcriptome profiles of a gas-fermenting bacterium

abstract:
Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. This rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation such as ribosomal RNA (rRNA) depletion and first-strand cDNA synthesis are tailored to a specific group of organisms (e.g. eukaryotes vs. prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products by NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand-specificity, and, most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies.

ImportanceRNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. Our work is important as we show that RNA-seq library preparation kits can strongly affect the outcome of the RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investment infeasible to most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting of sample processing workflows for any given organism.

category: systems biology


title: Sensory regulation of absence seizures in a mouse model of Gnb1 encephalopathy

abstract: Absence seizures, a type of non-convulsive epilepsy manifested by spike-wave discharges (SWD) in the electroencephalogram (EEG), display synchronous reciprocal excitation between the neocortex and thalamus. Recent studies have revealed that inhibitory neurons in the reticular thalamic (RT) nucleus and excitatory thalamocortical (TC) neurons are two key subcortical players in generating SWD. However, the signals that drive SWD-related thalamic activity remain elusive. Here, we show that SWD predominately occurs during wakefulness in several mouse models of absence epilepsy. In more focused studies of Gnb1 mutant mice, we found that sensory input regulates SWD. Using in vivo recording, we demonstrate that TC cells are activated prior to the onset of SWD and then inhibited during SWD. On the contrary, RT cells are slightly inhibited prior to SWD, but are strongly activated during SWD. Furthermore, chemogenetic activation of TC cells leads to the enhancement of SWD in Gnb1 mice. Together, our results indicate that sensory input in the periphery can regulate SWD by activating the thalamocortical pathway.


title: Human anelloviruses produced by recombinant expression of synthetic genomes.

abstract:
Human anelloviruses are acquired universally in infancy, highly prevalent, abundant in blood, and extremely diverse. Their apparent lack of pathogenicity indicates that they are a major component of the commensal human virome. Despite their being extensively intertwined with human biology, these viruses are poorly understood. A major impediment in studying anelloviruses is the lack of an in vitro system for their production and/ or propagation. Here we show that the T cell-derived human cell line MOLT-4 can be transfected with plasmids comprising tandem anellovirus genomes to produce viral particles visualized by electron microscopy. We found that a previously described human anellovirus of the Betatorquevirus genus (LY2), as well as a second Betatorquevirus detected by sequencing DNA extracted from a human retinal pigmental epithelium (nrVL4619), can be synthesized and produced by these means, enabling further molecular virology studies. Southern blot was used to demonstrate replication, and site-directed mutagenesis of the viral genome was performed to show that the production of anellovirus in this cell line is dependent on the expression of certain viral proteins. Finally, experiments performed in mice using purified nrVL4619 particles produced in MOLT-4 cells demonstrated infectivity in vivo in the tissue of origin. These results indicate that anelloviruses can be produced in vitro and manipulated to improve our understanding of this viral family which is ubiquitous in humans and many other mammals. Applications of this work to gene therapy and other therapeutic modalities are currently under investigation.

IMPORTANCEAnelloviruses are a major component of the human virome. However, their biology is not well understood mainly due to the lack of an in vitro system for anellovirus production and/or propagation. In this study, we used multiple orthogonal measures to show that two different anelloviruses belonging to the Betatorquevirus genus can be produced in a T-cell-derived human cell line, MOLT-4, via recombinant expression of synthetic genomes. Additionally, we show that anellovirus particles generated in this in vitro system demonstrate infectivity in vivo. Our findings enable new molecular virology studies of this highly prevalent, non-pathogenic, and weakly immunogenic family of viruses, potentially leading to therapeutic applications.


title: MYXOBACTERIA FROM VIETNAM: ISOLATION, PHYLOGENETIC ANALYSIS AND BIOPROSPECTION

abstract: Forty-three myxobacterial strains were isolated and identified from 20 soil samples collected in Vietnam. The information on morphological characteristics and 16S ribosomal gene sequencing showed that these strains were designated to seven genera belonging to Angiococcus, Archangium, Chondromyces, Corallococcus, Cystobacter, Melittangium, and Myxococcus, in suborder Cystobacterineae and Sorangiineae. The phylogenetic tree was constructed to clarify the genetic relationship between myxobacterial isolates. Myxobacteria were cultured, and crude extracts were obtained after 10-day fermentation in P-medium in the presence of the Amberlite XAD 16N adsorbent resin. Elution was carried out with acetone and methanol to obtain the crude extracts. Evaluation of antioxidant activity used the DPPH and ABTS assay, the minimum inhibitory concentration values were determined by the microdilution method. The total extract from CT21 had the highest total antioxidant activity (IC50 = 52.34 {+/-} 1.47 {micro}g/mL, 30.28 {+/-} 0.74 {micro}g/mL for DPPH and ABTS assays, respectively). The other potential strain was TG131 and GL41 that IC50 values were 40.28 {+/-} 1.13 and 57.24 {+/-} 1.52 {micro}g/mL, respectively (by the DPPH method), and 48.35 {+/-} 0.58 and 42.76 {+/-} 0.50 {micro}g/mL, respectively (by the ABTS method). Interestingly, 100% isolated myxobacterial strains show inhibitory activity against at least one of the tested microorganisms. The potential antimicrobial strain was GL41, which inhibited all tested microorganisms, and the MIC values were 1 {micro}g/mL against MRSA, MSSA, S. faecalis, C. albicans, and A. niger. The highest active strains were members of Myxococcus sp. genus.


title: Cytoplasmic processing of human transfer RNAs

abstract: Biogenesis of different classes of eukaryotic RNAs proceeds via different pathways that require strict spatiotemporal resolution. The intracellular organization has evolved to provide order for RNA processing to coordinate different maturation steps with specific enzymatic reaction. In higher eukaryotes, processing of transfer RNAs (tRNAs) is postulated to be almost entirely intranuclear, while in lower eukaryotes like yeast, tRNA maturation is both nuclear and cytoplasmic. Here, we show that tRNA processing is largely cytosolic event in human cells. After transcription, unprocessed precursors of tRNAs (pre-tRNAs) bound by La protein are exported from the nucleus into the cytoplasm. Using cell fractionation analysis and pre-tRNA-specific fluorescence in situ hybridization (FISH) protocol, we show tRNA splicing/ligation and end-processing takes place in the cytoplasm where majority of processing enzymes is also located. We propose here a model where processing of intron-less and intron-containing pre-tRNAs is cytoplasmic similarly to the observed in lower eukaryotes, although tRNA splicing precedes 5- and 3-end processing in human cells unlike in yeast.


title: Variation in fine scale recombination rate in temperature-evolved Drosophila melanogaster populations in response to selection.

abstract: Meiotic recombination plays a critical evolutionary role in maintaining fitness in response to selective pressures due to changing environments. Variation in recombination rate has been observed amongst and between species and populations and within genomes across numerous taxa. Studies have demonstrated a link between changes in recombination rate and selection but the extent to which fine scale recombination rate varies between evolved populations during the evolutionary period in response to selection is under active research. Here we utilize a set of three temperature-evolved Drosophila melanogaster populations that were shown to have diverged in several phenotypes including recombination rate based on the temperature regime in which they evolved. Using whole genome sequencing data of these populations, we generated fine scale recombination maps of the three populations. We compare recombination rates and patterns among the three populations and show that they have diverged at fine scales but are conserved at broader scales. We further demonstrate a correlation between recombination rates and genomic variation in the three populations and observe variation in putative warm-spots between the populations with these enhanced areas and associated genes overlapping areas previously shown to have diverged in the three populations due to selection. These data support the existence of recombination modifiers in these populations which are subject to selection during evolutionary change.


title: Nanoparticle coatings for controlled release of quercetin from an angioplasty balloon

abstract: Peripheral artery disease (PAD) is a systemic vascular disease of the legs that results in a blockage of blood flow from the heart to the lower extremities. Now one of the most common causes of mortality in the U.S., the first line of therapy for PAD is to mechanically open the blockages using balloon angioplasty. Coating the balloons with antiproliferative agents can potentially reduce vessel re-narrowing, or restenosis after surgical intervention, but current drug-coated balloons releasing chemotherapy agents like paclitaxel have in some cases shown increased mortality long-term. Our aim was to design a novel drug-coated balloon using a polymeric nanodelivery system for a sustained release of polyphenols that reduce restenosis but with reduced toxicity compared to chemotherapy agents. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles with entrapped quercetin, a dimethoxy quercetin (rhamnazin), as well as quercetin conjugated to PLGA, were developed. Balloon catheters were coated with polymeric nanoparticles using an ultrasonic method, and nanoparticle characteristics, drug loading, coating uniformity and drug release were determined. The adhesion of nanoparticles to vascular smooth muscle cells and the antiproliferative effect of nano-delivered polyphenols were also assessed. Of the nanoparticle systems tested, those with covalently attached quercetin provided the most sustained release over a 6-day period. Although these particles adhered to cells to a smaller extent compared to other nanoparticle formulations, their attachment was resistant to washing. These particles also exhibited the greatest anti-proliferative effect. In addition, their attachment was not altered when the cells were grown in calcifying conditions, and in PAD tissue calcification is typically a condition that impedes drug delivery. Moreover, the ultrasonic coating method generated a uniform balloon coating. The polymeric nanoparticle system with covalently attached quercetin developed herein is thus proposed as a promising platform to reduce restenosis post-angioplasty.

category: pharmacology and toxicology


title: Balancing selection on genomic deletion polymorphisms in humans

abstract: A key question in biology is why genomic variation persists in a population for extended periods. Recent studies have identified examples of genomic deletions that have remained polymorphic in the human lineage for hundreds of millennia, ostensibly owing to balancing selection. Nevertheless, genome-wide investigations of ancient and possibly adaptive deletions remain an imperative exercise. Here, we used simulations to show an excess of ancient allele sharing between modern and archaic human genomes that cannot be explained solely by introgression or ancient structure under neutrality. We identified 63 deletion polymorphisms that emerged before the divergence of humans and Neanderthals and are associated with GWAS traits. We used empirical and simulation-based analyses to show that the haplotypes that harbor these functional ancient deletions have likely been evolving under time- and geography-dependent balancing selection. Collectively, our results suggest that balancing selection may have maintained at least 27% of the functional deletion polymorphisms in humans for hundreds of thousands of years.


title: Pubertal Development Underlies Optimization of Inhibitory Control Through Specialization of Ventrolateral Prefrontal Cortex

abstract: Inhibitory control improves into young adulthood after specialization of relevant brain systems during adolescence. However, the biological mechanisms supporting this unique transition are not well understood. Given that adolescence is defined by pubertal maturation, we examined relative contributions of chronological age and pubertal maturation to inhibitory control development. 105 8-19-year-olds completed 1-5 longitudinal visits (227 visits total) in which pubertal development was assessed via self-reported Tanner stage and inhibitory control was assessed with an in-scanner antisaccade task. As expected, percent and latency of correct antisaccade responses improved with age and pubertal stage. When controlling for pubertal stage, chronological age was distinctly associated with correct response rate. In contrast, pubertal stage was uniquely associated with antisaccade latency even when controlling for age. Chronological age was associated with fMRI task activation in several regions including the right dorsolateral prefrontal cortex, while puberty was associated with right ventrolateral prefrontal cortex (VLPFC) activation. Furthermore, task-related connectivity between VLPFC and cingulate was associated with both pubertal stage and response latency. These results suggest that while age-related developmental processes may support maturation of brain systems underlying the ability to inhibit a response, puberty may play a larger role in the effectiveness of generating cognitive control responses.


title: Joint effect of temperature and insect chitosan on the heat resistance of Bacillus cereus spores in rice derivatives

abstract:
The heat resistance of Bacillus cereus spores inoculated in a rice substrate supplemented with insect chitosan as an alternative antimicrobial was studied. Two concentrations of insect chitosan were considered in order to assess the role of the insect chitosan concentration during the heat process as an antimicrobial replacement for crustacean chitosan.

Results of the study indicated that the DT values were clearly higher in the substrate without chitosan than in the substrate containing chitosan thus indicating a greater heat resistance to heat treatment of the microorganism inoculated in the substrate without chitosan. This behaviour was also evidenced in the survival curves. There were no great differences between either of the insect chitosan concentrations tested regarding the DT values. The z values were 9.8 {o}C on rice substrate. 8.9 {o}C on rice substrate supplemented with insect chitosan at 150 {micro}g/mL and 10.7 {o}C on rice substrate supplemented with 250 {micro}g/mL of insect chitosan, the chitosan concentration appears to affect the z value of the microorganism. Our results indicate that the combination of heat with insect chitosan as an antimicrobial on foodstuffs subjected to cooking is feasible and can improve the safety of rice derivatives.


title: Snip1-PRC2 governs the intrinsic apoptosis program in the developing brain

abstract: Brain development requires the intricate balance between division, death, and differentiation of neural progenitor cells (NPCs). Here, we report the discovery of Snip1 as a key regulator of NPC self-renewal and death. In the embryonic brain, Snip1 depletion causes brain dysplasia with robust induction of apoptosis. In culture, Snip1-depleted NPCs had reduced self-renewing property. Snip1 protein binds to promoters and regulates the expression of genes involved in intrinsic apoptosis, cell division, and cortical development. The depletion of a key chromatin modifier Polycomb Repressive Complex 2 (PRC2) is sufficient to reduce apoptosis and partially rescue the development of the Snip1-depleted brain. PRC2 controls NPC expansion, brain regionalization, and cell fate specification by depositing H3K27me3 and suppressing transcription by RNA polymerase II. Our findings suggest that Snip1 exerts loci-dependent regulation of PRC2 and H3K27me3 to toggle between death, survival, and self-renewal in the developing brain.


title: Evolutionary processes, not environmental drivers, determine the resilience of natural populations

abstract: Escalating climatic and anthropogenic pressures expose ecosystems worldwide to increasingly frequent disturbances. Yet, our ability to forecast the responses of natural populations to global change is impeded by a limited understanding of how exposure to environmental change shapes population resilience. Here, we test the association between local regimes of environmental change and the resilience to disturbance of 2,242 natural populations across 369 terrestrial and aquatic animal and plant species. Unexpectedly, we find that recent abiotic disturbance regimes (<50 years) do not predict the inherent ability of populations to resist and recover from disturbances. Instead, population resilience carries a strong evolutionary signal, with survival and developmental investments playing a key role in shaping population responses to disturbance. Contrary to assumptions that exposure to more frequent disturbances confers a greater ability to cope with future global change, population resilience relies on evolutionary processes that have shaped species characteristics over many generations.

category: ecology


title: Control becomes habitual early on when learning a novel motor skill

abstract: When people perform the same task repeatedly, their behavior becomes habitual, or inflexible to changes in the goals or structure of a task. While habits have been hypothesized to be a key aspect of motor skill acquisition, there has been little empirical work investigating the relationship between skills and habits. To better understand this relationship, we examined whether and when peoples behavior would become habitual as they learned a challenging new motor skill. After up to ten days of practice, we altered the structure of the task to assess whether participants would flexibly adjust their behavior or habitually persist in performing the task the way they originally learned. We found that participants behavior became habitual early in practice--after only two days--at which point they were still relatively unskilled. These data demonstrate that motor skills become habitual after relatively little training, but can nevertheless further improve with practice.


title: Cortex-wide fast activation of VIP-expressing inhibitory neurons by reward and punishment

abstract: Reward and punishment powerfully inform ongoing behaviors and drive learning throughout the brain, including neocortex. Yet it remains elusive how these global signals are represented and impact local cortical computations. Previously we found that in auditory cortex, VIP-expressing interneurons are recruited by reinforcement feedback. Here, we used 3D random-access two-photon microscopy and fiber photometry to monitor VIP neural activity in dozens of cortical areas while mice learned an auditory decision task. We show that reward and punishment evoke a rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode of VIP interneurons showed variations in temporal dynamics in individual neurons and across areas. Neither their weak sensory tuning in visual cortex, nor their arousal state modulation was predictive of reinforcer responses of VIP interneurons. We suggest that VIP-expressing cortical inhibitory neurons transduce global reinforcement signals to provide disinhibitory control over local circuit computations and their plasticity.


title: CaV3.1 T-type calcium channels regulate spatial memory processing in the dorsal subiculum

abstract: The dorsal subiculum (dSub) is one of the key structures responsible for the formation of hippocampal memory traces but the contribution of individual ionic currents to its cognitive function is not well studied. Although we recently reported that low-voltage-activated T-type calcium channels (T-channels) are crucial for the burst firing pattern regulation in the dSub pyramidal neurons, their potential role in learning and memory remains unclear. Here we used in vivo local field potential recordings and miniscope calcium imaging in freely behaving mice coupled with pharmacological and genetic tools to address this gap in knowledge. We show that the CaV3.1 isoform of T-channels is critically involved in controlling neuronal activity in the dSub in vivo. Altering burst firing pattern by inhibiting T-channel activity markedly affects calcium dynamics, synaptic plasticity, neuronal oscillations and phase-amplitude coupling in the dSub, thereby disrupting spatial learning. These results provide a crucial causative link between the CaV3.1 channels, burst firing activity of dSub neurons and memory processing, thus further supporting the notion that changes in neuronal excitability regulate memory trace formation. We posit that subicular CaV3.1 T-channels could be a promising novel drug target for cognitive disorders.


title: Caveolae couple mechanical stress to integrin recycling and activation

abstract: Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active {beta}1-integrin content on the surface of Cav1KO fibroblasts. FRAP analysis and endocytosis/recycling assays revealed that active {beta}1-integrin is mostly endocytosed through the CLIC/GEEC pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4-dependent manner. Moreover, the threshold for PM tension-driven {beta}1-integrin activation is lower in Cav1KO MEFs than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.

category: cell biology


title: Environmental DNA from archived leaves reveals widespread temporal turnover and biotic homogenization in forest arthropod communities

abstract: A major limitation of current reports on insect declines is the lack of standardized, long-term, and taxonomically broad time series. Here, we demonstrate the utility of environmental DNA from archived leaf material to characterize plant-associated arthropod communities. We base our work on several multi-decadal leaf time series from tree canopies in four land use types, which were sampled as part of a long-term environmental monitoring program across Germany. Using these highly standardized and well-preserved samples, we analyze temporal changes in communities of several thousand arthropod species belonging to 23 orders using metabarcoding and quantitative PCR. Our data do not support widespread declines of -diversity or genetic variation within sites. Instead, we find a gradual community turnover, which results in temporal and spatial biotic homogenization, across all land use types and all arthropod orders. Our results suggest that insect decline is more complex than mere -diversity loss, but can be driven by {beta}-diversity decay across space and time.


title: ZW sex-chromosome evolution and contagious parthenogenesis in Artemia brine shrimp

abstract: Eurasian brine shrimp (genus Artemia) have closely related sexual and asexual lineages of parthenogenetic females, which produce rare males at low frequencies. Although they are known to have ZW chromosomes, these are not well characterized, and it is unclear whether they are shared across the clade. Furthermore, the underlying genetic architecture of the transmission of asexuality, which can occur when rare males mate with closely related sexual females, is not well understood. We produced a chromosome-level assembly for the Eurasian species A. sinica and characterized in detail the pair of sex chromosomes of this species. We combined this with short-read genomic data for the sexual species A. sp. Kazakhstan and several lineages of A. parthenogenetica, allowing us to perform a first in-depth characterization of sex-chromosome evolution across the genus. We identified a small differentiated region of the ZW pair that is shared by all sexual and asexual lineages, supporting the shared ancestry of the sex chromosomes. We also inferred that recombination suppression has spread to larger sections of the chromosome independently in the American and Eurasian lineages. Finally, we took advantage of a rare male, which we backcrossed to sexual females, to explore the genetic basis of asexuality. Our results suggest that parthenogenesis may be partly controlled by a locus on the Z chromosome, highlighting the interplay between sex determination and asexuality.


title: Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra- hepatic envrionment

abstract: Hepatitis B surface antigen (HBsAg) loss or functional cure (FC), is considered the desirable therapeutic outcome for chronic hepatitis B (CHB) patients. However, the immuno-pathological biomarkers and underlying mechanisms remain unclear. Here we present a comprehensive single cell-transcriptomic atlas together with immune-phenotyping of disease-associated cell states (DACS) isolated from intra-hepatic tissue and matched PBMCs of either CHB or FC patients. We find that the intra-hepatic environment displays specific cell identities and molecular signatures that are distinct from PBMCs. FC is associated with emergence of an altered adaptive immune response marked by CD4 cytotoxic T lymphocytes (CD4-CTLs), and an activated innate response represented by liver-resident natural killer (LR-NK) cells. Overall, these findings provide novel insights into immuno-pathological cell states associated with FC that could serve as prognostic biomarkers.


title: Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment


title: Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley

abstract: Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host nucleotide-binding leucine-rich-repeat (NLR) receptors. The barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley NLR, MLA1, and therefore designated AVRA1. Here we show that AVRA1 and the sequence-unrelated Bgh effector BEC1016 (CSEP0491) suppress immunity in barley cells. We then used yeast 2-hybrid next-generation-interaction screens (Y2H-NGIS), followed by binary Y2H and bimolecular fluorescence complementation, to identify a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein, HvERdj3B. This is an ER protein quality control (ERQC) protein and silencing it increased the Bgh penetration rate in barley. HvERdj3B is localized to the ER lumen, and AVRA1 and BEC106 translocation into the ER was confirmed using a split GFP system. Together, these results suggest that the barley innate immunity, preventing Bgh entry into epidermal cells, is dependent on ERQC, which in turn is regulated by J-domain protein HvERdj3B and the two effectors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor, MLA1. We speculate whether the AVRA1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition.

category: plant biology


title: CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue

abstract:
Immunohistochemistry on archival human brains is often limited because several conditions arise that complicate the use for high-resolution fluorescence microscopy. In this study, we developed a novel clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed hCLARITY. hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of the Alzheimers disease were preserved with hCLARITY, and importantly classical DAB or Nissl stainings are compatible with this protocol. hCLARITY is extremely versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labelling approaches, e.g., in super-resolution microscopy. Taken together, hCLARITY enables research of the human brain with highest sensitivity and down to sub-diffraction resolution and therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases.

Striking Image / Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/489700v1_ufig1.gif" ALT="Figure 1">
View larger version (79K):
org.highwire.dtl.DTLVardef@1732df2org.highwire.dtl.DTLVardef@cda615org.highwire.dtl.DTLVardef@152b44aorg.highwire.dtl.DTLVardef@ba972a_HPS_FORMAT_FIGEXP  M_FIG Summary of the main advantages of hCLARITY. CLARITY was applied on human post mortem brain tissue in direct comparison to untreated sections (minus CLARITY). Owing to removal of lipids during the clearing with SDS and the resulting reduction in light scattering, cleared sections were less opaque. The denaturing effect of the detergent SDS most likely results in better accessibility of certain epitopes. The major benefits of hCLARITY were found in an increased sensitivity and specificity of antibodies for neuronal cells. The adapted hCLARITY protocol is compatible with staining techniques for confocal microscopy, super-resolution microscopy, and light microscopy.

C_FIG


title: Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

abstract: Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2, but not Munc13-1, traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of synaptotagmin-7 (syt7). However, in the absence of syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2 driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends obligatorily on functional pairing with syt7. Overall, DAG/phorbolester, ubMunc13-2 and syt7 form a stimulatory triad for dense-core vesicle priming.


title: Centrally expressed Cav3.2 T-type calcium channel is critical for the initiation and maintenance of neuropathic pain

abstract: Cav3.2 T-type calcium channel is a major molecular actor of neuropathic pain in peripheral sensory neurons, but its involvement at the supra-spinal level is almost unknown. In the Anterior Pretectum (APT), a hub of connectivity of the somatosensory system involved in pain perception, we show that Cav3.2 channels are expressed in a sub-population of GABAergic neurons co-expressing parvalbumin (PV). In these PV-expressing neurons, Cav3.2 channels contribute to a high frequency bursting activity, which is increased in the spared nerve injury model of neuropathy. Specific deletion of Cav3.2 channels in APT neurons reduced both the initiation and maintenance of mechanical and cold allodynia. These data are a direct demonstration that centrally expressed Cav3.2 channels also play a fundamental role in pain pathophysiology.