title: Critical Negatively Charged Residues Are Important for the Activity of SARS-CoV-1 and SARS-CoV-2 Fusion Peptides

abstract: Coronaviruses are a major infectious disease threat, and include the human pathogens of zoonotic origin SARS-CoV ("SARS-1"), SARS-CoV-2 ("SARS-2") and MERS-CoV ("MERS"). Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified that the domain immediately downstream of the S2 cleavage site is the bona fide FP (amino acids 798-835) for SARS-1 using ESR spectroscopy technology. We also found that the SARS-1 FP induces membrane ordering in a Ca2+ dependent fashion. In this study, we want to know which residues are involved in this Ca2+ binding, to build a topological model and to understand the role of the Ca2+. We performed a systematic mutation study on the negatively charged residues on the SARS-1 FP. While all six negatively charged residues contributes to the membrane ordering activity of the FP to some extent, D812 is the most important residue. We provided a topological model of how the FP binds Ca2+ ions: both FP1 and FP2 bind one Ca2+ ion, and there are two binding sites in FP1 and three in FP2. We also found that the corresponding residue D830 in the SARS-2 FP plays a similar critical role. ITC experiments show that the binding energies between the FP and Ca2+ as well as between the FP and membranes also decreases for all mutants. The binding of Ca2+, the folding of FP and the ordering activity correlated very well across the mutants, suggesting that the function of the Ca2+ is to help to folding of FP in membranes to enhance its activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S proteins in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering as the separate FPs do, and the mutations of the negatively charged residues also greatly reduce the membrane ordering activity. However, the difference in kinetic between the PP and FP indicates a possible role of FP trimerization. This finding could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel to combat the ongoing COVID-19 pandemic.


title: A naturally DNase-free CRISPR-Cas12c enzyme silences gene expression

abstract: Used widely for genome editing in human cells, plants and animals, CRISPR-Cas enzymes including Cas9 and Cas12 provide RNA-guided immunity to microbes by targeting foreign DNA sequences for cleavage. We show here that the native activity of CRISPR-Cas12c protects bacteria from phage infection by binding to DNA targets without cleaving them, revealing that antiviral interference can be accomplished without chemical attack on the invader or general metabolic disruption in the host. Biochemical experiments demonstrate that Cas12c is a site-specific ribonuclease capable of generating mature CRISPR RNAs (crRNAs) from precursor transcripts. Furthermore, we find that crRNA maturation is essential for Cas12c-mediated DNA targeting. Surprisingly, however, these crRNAs direct double-stranded DNA binding by Cas12c using a mechanism that precludes DNA cutting. Cas12cs RNA-guided DNA binding activity enables robust transcriptional repression of fluorescent reporter proteins in cells. Furthermore, this naturally DNase-free Cas12c enzyme can protect bacteria from lytic bacteriophage infection when targeting an essential phage gene. Together these results show that Cas12c employs targeted DNA binding to provide anti-viral immunity in bacteria, providing a native DNase-free pathway for transient antiviral immunity.

category: biochemistry


title: Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

abstract: Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.


title: Interactions of SARS-CoV-2 protein E with cell junctions and polarity PDZ-containing proteins

abstract: The C-terminus of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein E contains a PBM (PDZ binding motif) targeting PDZ (PSD-95/Dlg/ZO-1) domains identical to the PBM of SARS-CoV. The latter is involved in the pathogenicity of the virus. Recently, we identified ten human PDZ-containing proteins showing significant interactions with SARS-CoV-2 protein E PBM. We selected several of them involved in cellular junctions and cell polarity (TJP1, PARD3, MLLT4, LNX2) and MPP5/Pals1 previously shown to interact with SARS-CoV E PBM. Targeting cellular junctions and polarity components is a common strategy by viruses to hijack cell machinery to their advantage. In this study, we showed that these host PDZ domains TJP1, PARD3, MLLT4, LNX2 and MPP5/PALS1 interact in a PBM-dependent manner in vitro and colocalize with the full-length E protein in cellulo, sequestrating the PDZ domains to the Golgi compartment. We solved three crystal structures of complexes between human LNX2, MLLT4 and MPP5 PDZs and SARS-CoV-2 E PBM highlighting its binding preferences for several cellular targets. Finally, we showed different affinities for the PDZ domains with the original SARS-CoV-2 C-terminal sequence containing the PBM and the one of the beta variant that contains a mutation close to the PBM. The acquired mutations in E protein localized near the PBM might have important effects both on the structure and the ion-channel activity of the E protein and on the host machinery targeted by the variants during the infection.


title: Endowing Universal CAR T-cell with Immune-Evasive Properties using TALEN-Gene Editing.

abstract: Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive CAR T-cells scaffold that evades NK cell and alloresponsive T-cell attacks and imparts efficient antitumor activity in vitro and in vivo. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for future powerful clinical applications.

category: immunology


title: IHF regulates host colonization factors in the bee gut symbiont Frischella perrara

abstract: Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The protobacterium Frischella perrara colonizes the hindgut of honey bees, where it causes a characteristic immune response leading to the formation of the scab phenotype. Genetic determinants required for the establishment of this specific association are unknown. Here, we isolated three point mutations in the genes encoding the DNA-binding protein integration host factor (IHF). All three mutants formed larger colonies than the wild type in vitro and did not produce an aryl polyene metabolite, which confers a yellow color to F. perrara colonies. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara and abolished scab development. Using RNAseq we find that IHF affects the expression of potential colonization factors, including a colibactin biosynthetic gene cluster, two Type 6 secretion systems, pili genes, and the aryl polyene biosynthesis pathway. Gene deletions of these components revealed different colonization defects. While some mutants were abolished in their capacity to colonize the gut, others colonized but did not trigger the scab phenotype. IHF is conserved across many bacteria and may thus regulate host colonization also in other animal symbionts.

category: microbiology


title: IL-21 signaling promotes early dissemination of KSHV infection in B lymphocytes

abstract: Factors influencing Kaposis sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Here, using our unique model of KSHV infection in tonsil lymphocytes, we investigate the influence of host cytokines on the establishment of KSHV infection in human B cells. Our data demonstrate that KSHV manipulates the host cytokine microenvironment during early infection and susceptibility is generally associated with downregulation of multiple cytokines. However, we show that IL-21 signaling promotes KSHV infection by promoting both plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. Our data reveal that this phenotype is dependent upon a specific milieu of T cells, that includes IL-21 producing Th17, Tc17 and CD8+ central memory T cells. These results suggest that IL-21 plays a significant role in the early stages of KSHV infection in the human immune system and that specific immunological states favor the initial establishment of KSHV infection by increasing infection in plasma cells.


title: IL-21 signaling promotes the establishment of KSHV infection in human tonsil lymphocytes by increasing early targeting of plasma cells


title: Design principles to assemble drug combinations for effective tuberculosis therapy using interpretable pairwise drug response measurements

abstract: A challenge in designing treatment regimens for tuberculosis is the necessity to use three or more antibiotics in combination. The combination space is too large to be comprehensively assayed; therefore, only a small number of possible combinations are tested. We narrowed the prohibitively large search space of combination drug responses by breaking down high-order combinations into units of drug pairs. Using pairwise drug potency and drug interaction metrics from in vitro experiments across multiple growth environments, we trained machine learning models to predict outcomes associated with higher-order combinations in the BALB/c relapsing mouse model, an important preclinical model for drug development. We systematically predicted treatment outcomes of >500 combinations among twelve antibiotics. Our classifiers performed well on test data and predicted many novel combinations to be improved over bedaquiline + pretomanid + linezolid, an effective regimen for multidrug-resistant tuberculosis that also shortens treatment in BALB/c mice compared to the standard of care. To understand the design features of effective drug combinations, we reformulated classifiers as simple rulesets to reveal guiding principles of constructing combination therapies for both preclinical and clinical outcomes. One example ruleset is to include a drug pair that is synergistic in dormancy and another pair that is potent in a cholesterol-rich growth environment. These rulesets are predictive, intuitive, and practical, thus enabling rational construction of effective drug combinations based on in vitro pairwise drug synergies and potencies. As more preclinical and clinical drug combination data become available, we expect to improve predictions and combination design rules.


title: Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion.

abstract:
Salmonella enterica serovar Typhimurium (Salmonella) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, Salmonella produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species and the Salmonella genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during Salmonella pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. Salmonella lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient Salmonella strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed Salmonella dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type Salmonella, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of Salmonella chitinases. This analysis also revealed differential abundance of Lewis X-containing glycans that is likely a result of host cell modulation due to the detection of Salmonella chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that Salmonella chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.

Author SummarySalmonella Typhimurium infection is one of the leading causes of food-borne illnesses worldwide. In order for Salmonella to effectively cause disease, it has to invade the epithelial cells lining the intestinal tract. This invasion step allows Salmonella to replicate efficiently, causing further tissue damage and inflammation. In susceptible patients, Salmonella can spread past the intestines and infect peripheral organs. It is essential to fully understand the invasion mechanism used by Salmonella to design better treatments for infection. Here, we demonstrate that the two chitinases produced by Salmonella are involved in this invasion process. We show that Salmonella chitinases interact with surface glycans of intestinal epithelial cells and promote adhesion and invasion. Using a mouse infection model, we show that Salmonella chitinases are required for the invasion of the small intestine and enhance the dissemination of Salmonella to other organs. This study reveals an additional mechanism by which Salmonella invades and causes infection.


title: Polyunsaturated fatty acid arachidonic acid increases the virulence of gut pathogen, Yersinia enterocolitica

abstract: Food-borne illnesses are a major health concern worldwide. While 1 in 6 individuals are infected in the United States yearly, there is little research into which dietary factors can alter the risk of infection. Despite evidence suggesting a correlation between obesity and enteric infection, the few reported studies focus on the role of dietary factors and the impact on host tissues and susceptibility. The direct impact of dietary constituents on the virulence of a pathogen has largely been ignored. One component of the Western diet that has been correlated with increasing inflammatory diseases is increased consumption of omega-6 polyunsaturated fatty acids such as arachidonic acid. Here, we show that arachidonic acid directly alters the pathogenicity of the food-borne pathogen Yersinia enterocolitica. Using in vitro cellular adherence assays, proteomic peptide mass fingerprint profiles and in vivo mouse models, we show that arachidonic acid can alter the pathogenesis of Y. enterocolitica by increasing proliferation and intracellular invasion. These findings have major implications in more than food safety, potentially revealing how current dietary habits may increase the virulence of food-borne pathogens.


title: N-acetyl-cysteinylated streptophenazines fromStreptomyces

abstract:
Here, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times, but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example on how to leverage metabolomics for the discovery of new natural products and point out to the often-neglected contribution of house-keeping cellular functions to natural product diversification.

Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY


title: Predictions of the SARS-CoV-2 Omicron Variant (B.1.1.529) Spike Protein Receptor-Binding Domain Structure and Neutralizing Antibody Interactions

abstract: The genome of the SARS-CoV-2 Omicron variant (B.1.1.529) was released on November 22, 2021, which has caused a flurry of media attention due the large number of mutations it contains. These raw data have spurred questions around vaccine efficacy. Given that neither the structural information nor the experimentally-derived antibody interaction of this variant are available, we have turned to predictive computational methods to model the mutated structure of the spike proteins receptor binding domain and posit potential changes to vaccine efficacy. In this study, we predict some structural changes in the receptor-binding domain that may reduce antibody interaction without completely evading existing neutralizing antibodies (and therefore current vaccines).

category: molecular biology


title: A two-step metagenomics approach for prey identification from the blood meals of common vampire bats (Desmodus rotundus)

abstract: The feeding behaviour of the sanguivorous common vampire bat (Desmodus rotundus) facilitates the transmission of pathogens that can impact both human and animal health. To formulate effective strategies in controlling the spread of diseases, there is a need to obtain information on which animals they feed on. One DNA-based approach, shotgun sequencing, can be used to obtain such information. Even though it is costly, shotgun sequencing can be used to simultaneously retrieve prey and vampire bat mitochondrial DNA for population studies within one round of sequencing. However, due to the challenges of analysing shotgun sequenced metagenomic data such as false negatives/positives and typically low proportion of reads mapped to diet items, shotgun sequencing has not been used for the identification of prey from common vampire bat blood meals. To overcome these challenges and generate longer mitochondrial contigs which could be useful for prey population studies, we shotgun sequenced common vampire bat blood meal samples (n=8) and utilised a two-step metagenomic approach based on combining existing bioinformatic workflows (alignment and de novo mtDNA assembly) to identify prey. Further, we validated our results to detections made through metabarcoding. We accurately identified the common vampire bats prey in seven out of eight samples without any false positives. We also generated prey mitochondrial contig lengths between 138bp to 3231bp (mean=985bp, SD=981bp). As we develop more computationally efficient bioinformatics pipelines and reduce sequencing costs, we can expect an uptake in metagenomics dietary studies in the near future.


title: Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR- V7 splice variant chromatin binding and transcriptional activities

abstract: The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.


title: TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

abstract: Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

category: cell biology


title: THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

abstract: Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2{Delta} mutants. In contrast, CAG fragility and TRCs in the trf4{Delta} mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

category: genetics


title: Mathematical expressions describing enzyme velocity and inhibition at high enzyme concentration

abstract: Enzyme behaviour is typically characterised in the laboratory using very diluted solutions of enzyme. However, in vivo processes usually occur at [ST] {approx} [ET] {approx} Km. Furthermore, the study of enzyme action usually involves analysis and characterisation of inhibitors and their mechanisms. However, to date, there have been no reports proposing mathematical expressions that can be used to describe enzyme activity at high enzyme concentration apart from the simplest single substrate, irreversible case. Using a continued fraction approach, equations can be easily derived to apply to the most common cases in monosubstrate reactions, such as irreversible or reversible reactions and small molecule (inhibitor or activator) kinetic interactions. These expressions are simple and can be understood as an extension of the classical Michaelis-Menten equations. A first analysis of these expressions permits to deduce some differences at high vs low enzyme concentration, such as the greater effectiveness of allosteric inhibitors compared to catalytic ones. Also, they can be used to understand catalyst saturation in a reaction. Although they can be linearised following classical approaches, these equations also show some differences that need to be taken into account. The most important one may be the different meaning of line intersection points in Dixon plots. All in all, these expressions may be useful tools for the translation in vivo of in vitro experimental data or for modelling in vivo and biotechnological processes.


title: Creation of photocyclic vertebrate rhodopsin by single amino acid substitution

abstract: Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photo-convert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, the molecular property of vertebrate rhodopsin successfully converted to the photocyclic and photoreversible properties by a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by the regulation of the recovery from the active state to the original dark state.


title: FPCountR: Absolute quantification of fluorescent proteins for synthetic biology

abstract: This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe a novel  ECmax assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It also reveals that mCherry absorption at 600nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.

category: synthetic biology


title: ARBRE: Computational resource to predict pathways towards industrially important aromatic compounds

abstract: Synthetic biology and metabolic engineering rely on computational search tools for predictions of novel biosynthetic pathways to industrially important compounds, many of which are derived from aromatic amino acids. Pathway search tools vary in their scope of covered reactions and compounds, as well as in metrics for ranking and evaluation. In this work, we present a new computational resource called ARBRE: Aromatic compounds RetroBiosynthesis Repository and Explorer. It consists of a comprehensive biochemical reaction network centered around aromatic amino acid biosynthesis and a computational toolbox for navigating this network. ARBRE encompasses over 28000 known and 100000 novel reactions predicted with generalized enzymatic reactions rules and over 70000 compounds, of which 22000 are known to biochemical databases and 48000 only to PubChem. Over 1,000 molecules that were solely part of the PubChem database before and were previously impossible to integrate into a biochemical network are included into the ARBRE reaction network by assigning enzymatic reactions. ARBRE can be applied for pathway search, enzyme annotation, pathway ranking, visualization, and network expansion around known biochemical pathways to predict valuable compound derivations. In line with the standards of open science, we have made the toolbox freely available to the scientific community at http://lcsb-databases.epfl.ch/arbre/. We envision that ARBRE will provide the community with a new computational toolbox and comprehensive search tool to predict and rank pathways towards industrially important aromatic compounds.


title: Evaluation of odorant-binding protein-1 as a molecular marker for identifying biological forms and delimitating sibling species of Anopheles stephensi

abstract:
BackgroundAnopheles stephensi, an invasive malaria vector, has been reported to have three biological forms identifiable based on the number of ridges present on the eggs floats and the dimension of eggs. Recently, these forms have been designated as sibling species based on the fixed differences in the DNA sequence of the first intron of the odorant-binding protein-1 (AsteObp1). In this study, we evaluated the utility of this neutral marker in designating sibling species or identifying biological forms.

MethodsField collected and laboratory-reared An. stephensi were characterized for biological forms based on the number of floats on egg-ridge. DNA sequencing of the partial AsteObp1 gene of An. stephensi individuals were performed by Sangers method, either directly or after cloning with a plasmid vector.

ResultsAsteObp1 intron-1 in Indian An. stephensi populations are highly polymorphic with the presence of more than 12 haplotypes exhibiting nucleotide-as well as length-polymorphism (90-to-121 bp). A majority of the field samples were heterozygous (up to 89% in the field populations). The phasing of haplotypes in heterozygotes through Sangers sequencing was challenging due to indels (1-to-24 bp) at multiple loci. No specific haplotype or monophyletic clade of intron-1 was found associated with a specific biological form. The inbreeding coefficient for this marker was close to zero in field and laboratory populations which refute the existence of sibling species based on the AsteObp1 marker.

ConclusionsAsteObp1 cannot serve as a marker for the identification of biological forms of An. stephensi. The probable existence of sibling species in An. stephensi based on the AsteObp1 intron-1 is refuted.


title: Tailer: A Pipeline for Sequencing-Based Analysis of Non-Polyadenylated RNA 3' End Processing

abstract: Post-transcriptional trimming and tailing of RNA 3 ends play key roles in the processing and quality control of non-coding RNAs (ncRNAs). However, bioinformatic tools to examine changes in the RNA 3 "tailome" are sparse and not standardized. Here we present Tailer, a bioinformatic pipeline in two parts that allows for robust quantification and analysis of tail information from next generation sequencing experiments that preserve RNA 3 end information. The first part of Tailer, Tailer-Processing, uses genome annotation or reference FASTA gene sequences to quantify RNA 3 ends from SAM-formatted alignment files or FASTQ sequence read files produced from sequencing experiments. The second part, Tailer-Analysis, uses the output of Tailer-Processing to identify statistically significant RNA targets of trimming and tailing and create graphs for data exploration. We apply Tailer to RNA 3 end sequencing experiments from three published studies and find that it accurately and reproducibly recapitulates key findings. Thus, Tailer should be a useful and easily accessible tool to globally investigate tailing dynamics of non-polyadenylated RNAs and conditions that perturb them.


title: Ca2+ Mediates HIF-dependent Upregulation of Aquaporin 1 in Pulmonary Arterial Smooth Muscle Cells

abstract: Prolonged exposure to hypoxia causes structural remodeling and sustained contraction of the pulmonary vasculature, resulting in the development of pulmonary hypertension. Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration contribute to the vascular remodeling. We previously showed that the protein expression of aquaporin 1 (AQP1), a membrane water channel protein, is elevated in PASMCs during following in vivo or in vitro exposure to hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)-1. Moreover, we and others have shown that an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process occurred via transcriptional regulation or was Ca2+-dependent. PASMCs were exposed to hypoxia, incubated with DMOG, which inhibits HIF protein degradation or infected with constitutively active forms of HIF-1 or HIF-2. Hypoxia, DMOG and HIF1/2 produced a time-dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with increasing HIF1/2 levels and DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage-gated Ca2+ channel inhibitor, verapamil (VER) and nonselective cation channel inhibitor SKF96365 (SKF). VER and SKF also blocked upregulation of AQP1 protein by DMOG or HIF1/2, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Silencing of AQP1 abrogated increases in PASMC migration and proliferation induced by HIF1/2, suggesting induction of AQP1 protein by HIF1/2 has a functional outcome in these cells. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF-dependent increases in [Ca2+]i.


title: Genetic conversion of a split-drive into a full-drive element

abstract: Gene-drive systems offer an important new avenue for spreading beneficial traits into wild populations. Their core components, Cas9 and guide RNA (gRNA), can either be linked within a single cassette (full gene drive, fGD) or provided in two separate elements (split gene drive, sGD) wherein the gRNA-bearing element drives in the presence of an independent static source of Cas9. We previously designed a system engineered to turn split into full gene drives. Here, we provide experimental proof-of-principle for such a convertible system inserted at the spo11 locus, which is recoded to restore gene function. In multigenerational cage studies, the reconstituted spo11 fGD cassette initially drives with slower kinetics than the unlinked sGD element (using the same Mendelian vasa-Cas9 source), but eventually reaches a similar level of final introgression. Different kinetic behaviors may result from transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.


title: Immune disease risk variants regulate gene expression dynamics during CD4+ T cell activation

abstract: During activation, T cells undergo extensive changes in gene expression which shape the properties of cells to exert their effector function. Therefore, understanding the genetic regulation of gene expression during T cell activation provides essential insights into how genetic variants influence the response to infections and immune diseases. We generated a single-cell map of expression quantitative trait loci (eQTL) across a T cell activation time-course. We profiled 655,349 CD4+ naive and memory T cells, capturing transcriptional states of unstimulated cells and three time points of cell activation in 119 healthy individuals. We identified 38 cell clusters, including stable clusters such as central and effector memory T cells and transient clusters that were only present at individual time points of activation, such as interferon-responding cells. We mapped eQTLs using a T cell activation trajectory and identified 6,407 eQTL genes, of which a third (2,265 genes) were dynamically regulated during T cell activation. We integrated this information with GWAS variants for immune-mediated diseases and observed 127 colocalizations, with significant enrichment in dynamic eQTLs. Immune disease loci colocalized with genes that are involved in the regulation of T cell activation, and genes with similar functions tended to be perturbed in the same direction by disease risk alleles. Our results emphasize the importance of mapping context-specific gene expression regulation, provide insights into the mechanisms of genetic susceptibility of immune diseases, and help prioritize new therapeutic targets.

category: genomics


title: Functional Anatomy of the TDP-43 Redox Sensor

abstract: TAR binding protein 43 (TDP-43) is an RNA binding protein that assists in the maturation, export and sub-cellular localization of mRNA. The carboxyl terminal 153 residues of TDP-43 are of low sequence complexity and allow for self-association of the protein in a manner leading to its phase separation from an aqueous environment. These interactions assist TDP-43 in forming cytoplasmic RNA granules involved in the transport of mRNA for localized translation. Self-association of the TDP-43 low complexity (LC) domain is facilitated by a region of twenty five residues that are of extreme evolutionary conservation. The molecular basis for self-adherence of the protein through this region has been illuminated by a combination of structural and biochemical studies, allowing definition of a morphologically specific cross-{beta} structure predicted to be weakly assembled by main chain hydrogen bonds. In this study we have investigated the importance of individual, Pauling hydrogen bonds hypothesized to facilitate self-adherence of the TDP-43 LC domain.


title: Tracing the genetics of neurological disease to the mutation-directed addition of single hydrogen bonds

abstract: Mutations causative of neurological and neurodegenerative disease can occur in coding regions that specify protein domains of low sequence complexity. These autosomal dominant mutations can be idiosyncratic in their recurrent appearance at the same amino acid. Here we report studies of recurrent mutations in proline residues located within low complexity (LC) domains associated with the neurofilament light chain protein, the microtubule-associated tau protein, and the heterogeneous nuclear RNPA2 protein. All such mutations manifest their effects by directing formation of variant proteins endowed with the addition of a single, main chain hydrogen bond specified by the variant amino acid replacing proline. Here we show that methylation of the peptide backbone nitrogen atom associated with these variant amino acids eliminates the aberrant hydrogen bond and restores normal protein function.


title: SAVED by a toxin: Structure and function of the CRISPR Lon protease.

abstract: CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea 1, 2. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors 6. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack 7, 8. Among a large set of proteins that were predicted to be linked to the type III systems 9, 10, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED-instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.


title: Modeling the trajectory of SARS-CoV-2 spike protein evolution in continuous latent space using a neural network and Gaussian process

abstract: Viral vaccines can lose their efficacy as the genomes of targeted viruses rapidly evolve, resulting in new variants that may evade vaccine-induced immunity. This process is apparent in the emergence of new SARS-CoV-2 variants which have the potential to undermine vaccination efforts and cause further outbreaks. Predictive vaccinology points to a future of pandemic preparedness in which vaccines can be developed preemptively based in part on predictive models of viral evolution. Thus, modeling the trajectory of SARS-CoV-2 spike protein evolution could have value for mRNA vaccine development. Traditionally, in silico sequence evolution has been modeled discretely, while there has been limited investigation into continuous models. Here we present the Viral Predictor for mRNA Evolution (VPRE), an open-source software tool which learns from mutational patterns in viral proteins and models their most statistically likely evolutionary trajectories. We trained a variational autoencoder with real-time and simulated SARS-CoV-2 genome data from Australia to encode discrete spike protein sequences into continuous numerical variables. To simulate evolution along a phylogenetic path, we trained a Gaussian process model with the numerical variables to project spike protein evolution up to five months in advance. Our predictions mapped primarily to a sequence that differed by a single amino acid from the most reported spike protein in Australia within the prediction timeframe, indicating the utility of deep learning and continuous latent spaces for modeling viral protein evolution. VPRE can be readily adapted to investigate and predict the evolution of viruses other than SARS-CoV-2 in temporal, geographic, and lineage-specific pathways.

category: bioinformatics


title: Pandemic-scale phylogenetics

abstract: 1.Phylogenetics has been central to the genomic surveillance, epidemiology and contact tracing efforts during the COVD-19 pandemic. But the massive scale of genomic sequencing has rendered the pre-pandemic tools inadequate for comprehensive phylogenetic analyses. Here, we discuss the phylogenetic package that we developed to address the needs imposed by this pandemic. The package incorporates several pandemic-specific optimization and parallelization techniques and comprises four programs: UShER, matOptimize, RIPPLES and matUtils. Using high-performance computing, UShER and matOptimize maintain and refine daily a massive mutation-annotated phylogenetic tree consisting of all SARS-CoV-2 sequences available in online repositories. With UShER and RIPPLES, individual labs - even with modest compute resources - incorporate newly-sequenced SARS-CoV-2 genomes on this phylogeny and discover evidence for recombination in real-time. With matUtils, they rapidly query and visualize massive SARS-CoV-2 phylogenies. These tools have empowered scientists worldwide to study the SARS-CoV-2 evolution and transmission at an unprecedented scale, resolution and speed.


title: miR-199-3p Suppresses Cellular Migration and Viability and Promotes Progesterone Production in Goose Ovarian Follicles Before Selection by Targeting ITGB8 and Regulating Other ECM-related Genes

abstract: The extracellular matrix (ECM) constitutes the follicular basal lamina and is also present between follicular cells. Remodeling of the ECM is believed to be a key event in follicular development, especially follicular selection, and plays important roles in cell migration, survival, and steroidogenesis. miR-199-3p is differentially expressed in the goose follicular granulosa layer during follicular selection and is reported to play a primary role in inhibiting cell migration and invasion. Nevertheless, the effect of miR-199-3p on ovarian follicles and its role in follicular cellular migration are not understood. In this study, we demonstrated by qRT-PCR that miR-199-3p was differentially expressed in the granulosa layer from goose ovarian follicles before and after follicular selection. Additionally, we found that miR-199-3p overexpression could significantly suppress cell viability and migration, as well as elevate both the concentration of progesterone and the expression of key progesterone production genes in cultured granulosa cells (GCs) from goose pre-hierarchical follicles. Furthermore, using dual-fluorescence reporter experiments on 293T cells, we confirmed that miR-199-3p downregulated the expression of the ECM gene ITGB8 by directly targeting its mRNA three prime untranslated region (3' UTR). Finally, we found that miR-199-3p overexpression in the GCs of goose pre-hierarchical follicles inhibited the expression of two ECM-related genes (MMP9 and MMP15) yet promoted the expression of another two ECM-related genes (COL4A1 and LAMA1). Taken together, these findings suggest that miR-199-3p participates in granulosa cell migration, viability, and steroidogenesis in goose ovarian follicles before selection by targeting ITGB8 and modulating other ECM-related genes. These data highlight the key roles of miR-199-3p in follicular cell migration, viability, and steroidogenesis by regulating ECM-related genes and thus contribute to a better understanding of the mechanisms underlying follicle selection in birds.


title: Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers

abstract: High throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. Chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity. Here, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we identify and visualize both novel and previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin.


title: Deltamethrin contact exposure mediated toxicity and histopathological aberrations in tissue systems of cockroach species Periplaneta americana and Blatella germanica

abstract: Cockroach species Periplaneta americana and Blatella germanica potentially survive in locations close to human activity. Besides spoiling food material, cockroaches also transfer pathogens of different diseases among human. Since the insecticides have been used extensively to control cockroaches, information on their insecticide susceptibility and toxicity at cellular level may be crucial. In the study, deltamethrin toxicity as well as the deltamethrin-mediated cytomorphological changes in brain, ovary and midgut of the two important cockroach species has been assessed. Different concentrations [0.00025% (0.0025mg/ml), 0.0025% (0.025mg/ml), 0.025 (0.25mg/ml), 0.25% (2.5mg/ml), 0.5% (5mg/ml), 1% (10mg/ml)] of deltamethrin in acetone were used to expose test species in WHO bottle assay. Knockdown was recorded after 5 min interval while delayed mortality was observed after 24 hr. Brain, ovary and gut were dissected post 1 hr exposure and 24 hr holding (for 0.25%, 0.5% and 1% concentration), and tissues were processed for microscopic analysis. Deltamethrin exposed cockroaches and dissected tissues were used to estimate deltamethrin using HPLC. At 0.00025% (lowest concentration), the percentage knock-down observed was 66.7% for P. americana and 80% B. germanica respectively (R2= 0.78; p=0.0001) in 1 hr. KDT50 value was found to be 8.7 min (95% CI: 7.3-10.2), while KDT99 was 20.7 min (95% CI: 16.0-35.7) in P. americana at 1% concentration. Whereas, the KDT50 and KDT99 values for B. germanica were 7.4 min (95% CI: 5.4-9.1) and 27.4 min (95% CI: 18.2-80.0) at similar concentration. LD50 and LD95 values (for 60 min standard exposure) were 0.0006 % (95% CI: 0.00-0.001) and 0.034% (95% CI: 0.013-0.49) respectively for P. americana, while these values were 0.0005 (95% CI: 0.00-0.001) and 0.04 (95% CI: 0.01-0.23) for B. germanica. Exposure to 1% deltamethrin induced considerable toxic effect in the epithelial cells in the midgut. HPLC estimated 0.21{+/-}0.05 mg (95% CI - 0.18-0.25; CoV 23.9%) deltamethrin in P. americana post 1% exposure. Even short term exposure of low concentration of synthetic pyrethroid deltamethrin displayed immediate knockdown and delayed mortality in both the test species. Considerable histological damage was observed in both the insects at 1% exposure. In India, resistance to deltamethrin may have been reported among different insects due its extensive use, however the formulations such as insecticide paints, attractant baits etc. developed using deltamethrin as active ingredient could be useful in cockroach control operations.

category: zoology


title: Dairy vs. beef production - expert views on welfare

abstract: Consumers views and concerns about the welfare of farm animals may play an important role in their decision to consume dairy, meat and/or plants as their primary protein source. As animals are killed prematurely in both dairy and beef industries, it is important to quantify and compare welfare compromise in these two sectors before the point of death. Seventy world-leading bovine welfare experts based in 23 countries, were asked to evaluate the likelihood of a bovine to experience 12 states of potential welfare concern, inspired by the Welfare Quality(R) protocol. The evaluation focused on the most common beef and dairy production systems in the experts country, and was carried out separately for dairy/beef calves raised for red-meat, dairy/beef calves raised for veal, dairy/beef calves raised as replacement, and for dairy/beef cows. The results show experts rated the overall likelihood of a negative welfare state (i.e. welfare risk) to be higher in animals from dairy herds than from beef herds, for all animal categories, regardless of whether they were used to produce milk, red-meat or veal. These findings suggest that consuming food products derived from common dairy production systems (dairy or meat), may be more harmful to the welfare of animals than consuming products derived from common beef production systems (i.e. from animals solely raised for their meat). Raising awareness about the linkage between dairy and meat production, and the toll of milk production on the welfare state of animals in the dairy industry, may encourage a more sustainable and responsible food consumption.

category: animal behavior and cognition


title: The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes

abstract:
Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modification in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster.

Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24- mutant, as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic.

In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.


title: Escherichia coli antimicrobial resistance; Phenotypic and genotypic characterization

abstract: Improper use of the antimicrobials as E. coli giving the microorganism multi-resistance against many antimicrobials by gene mutation on integrons, transposons and plasmids. Therefore, our aim in this study is to 1)examine antibiotics resistance phenotype and genotype in Escherichia coli, 2) identifying the structure of bacterial resistance genes on whole-genome sequencing against multi-drug resistant of Escherichia coli in marketed poultry meat. Samples collected, prepared and Bacteriological examination, Antimicrobial sensitivity test performed, Serological identification of Escherichia coli isolates. Results declared that; the prevalence of E. coli from tested chicken meat samples of 100 chicken meat samples surveyed against E. coli the result declared that about 40%. Antimicrobial susceptibility was; antibiotics of choice against E. coli Sulfonamides, Cephalosporins,Tetracyclines, Quinolones. Serologically, STEC (O157:H7) 30%, ETEC (O142) 10%, EHEC (O26:H11). The subunit B of shiga-like toxin (SLT) gene appeared as a homogenous band. Heat-labile toxin (LT) gene was screened in both genomic DNA and plasmid preps in tested strains. Control STEC as it represents a danger to the poultry consumers. We recommended to increase the hygienic measures during slaughtering, processing and/or handling of chicken carcasses and avoidance unnecessary usage of any antimicrobials to avoid appearance of new antimicrobials resistant.


title: Sexual and developmental variations of ecto-parasitism in damselflies

abstract: The prevalence and intensity of parasitism can have different fitness costs between sexes, and across species and developmental stages. This variation could arise because of species specific sexual and developmental differences in body condition, immunity, and resistance. Theory predicts that the prevalence of parasitism will be greater in individuals with poor body condition and the intensity of parasitism will be greater in individuals with larger body size. These predictions have been tested and verified in vertebrates. In insects, however, contradictory evidence has been found in different taxa. Here, we tested these predictions on two species of Agriocnemis (Agriocnemis femina and Agriocnemis pygmaea) damselflies, which are parasitized by Arrenurus water mite ectoparasites. We measured body weight, total body length, abdomen area and thorax area of non-parasitized damselflies and found body condition varied between males and females, between immature females and mature females and between A. femina and A. pygmaea. Then, we calculated the parasite prevalence, i.e., the frequency of parasitism and intensity, i.e., the number of parasites per infected damselfly in eleven natural populations of both species. In line to our predictions, we observed greater prevalence in immature females than mature females but found no difference in parasite prevalence between males and females. Furthermore, we found that parasite load was higher in females than males and in immature females than mature females. Our result also showed that the frequency and intensity of parasitism varied between the two studied species, being higher in A. pygmaea than A. femina. Our study provides evidence that parasitism impacts sexes, developmental stages and species differentially and suggests that variation may occur due to sex, developmental stage, and species-specific resistance and tolerance mechanism.

category: evolutionary biology


title: Aggregation chimeras provide evidence of in vivo intercellular correction in ovine CLN6 neuronal ceroid lipofuscinosis (Batten disease)

abstract:
The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are fatal, mainly childhood, inherited neurodegenerative lysosomal storage diseases. Sheep affected with a CLN6 form display progressive regionally defined glial activation and subsequent neurodegeneration, indicating that neuroinflammation may be causative of pathogenesis. In this study, aggregation chimeras were generated from homozygous unaffected normal and CLN6 affected sheep embryos, resulting in seven chimeric animals with varied proportions of normal to affected cells. These sheep were classified as affected-like, recovering-like or normal-like, based on their cell-genotype ratios and their clinical and neuropathological profiles.

Neuropathological examination of the affected-like animals revealed intense glial activation, prominent storage body accumulation and severe neurodegeneration within all cortical brain regions, along with vision loss and decreasing intracranial volumes and cortical thicknesses consistent with ovine CLN6 disease. In contrast, intercellular communication affecting pathology was evident at both the gross and histological level in the normal-like and recovering-like chimeras, resulting in a lack of glial activation and rare storage body accumulation in only a few cells. Initial intracranial volumes of the recovering-like chimeras were below normal but progressively recovered to about normal by two years of age. All had normal cortical thicknesses, and none went blind. Extended neurogenesis was evident in the brains of all the chimeras.

This study indicates that although CLN6 is a membrane bound protein, the consequent defect is not cell intrinsic. The lack of glial activation and inflammatory responses in the normal-like and recovering-like chimeras indicate that newly generated cells are borne into a microenvironment conducive to maturation and survival.

category: neuroscience


title: Heterozygous transcriptional and nonsense decay signatures in blood outgrowth endothelial cells from patients with hereditary haemorrhagic telangiectasia

abstract: In order to identify cellular phenotypes resulting from nonsense (gain of stop/premature termination codon) variants, we devised a framework of analytic methods that minimised confounder contributions, and applied to blood outgrowth endothelial cells (BOECs) derived from controls and patients with heterozygous nonsense variants in ACVRL1, ENG or SMAD4 causing hereditary haemorrhagic telangiectasia (HHT). Following validation of 48 pre-selected genes by single cell qRT-PCR, discovery RNASeq ranked HHT-differential alignments of 16,807 Ensembl transcripts. Consistent gene ontology (GO) processes enriched compared to randomly-selected gene lists included bone morphogenetic protein, transforming growth factor-{beta} and angiogenesis GO processes already implicated in HHT, further validating methodologies. Additional terms/genes including for endoplasmic reticulum stress could be attributed to a generic process of inefficient nonsense mediated decay (NMD). NMD efficiency ranged from 78-92% (mean 87%) in different BOEC cultures, with misprocessed mutant protein production confirmed by pulse chase experiments. Genes in HHT-specific and generic nonsense decay (ND) lists displayed differing expression profiles in normal endothelial cells exposed to an additional stress of exogenous 10mol/L iron which acutely upregulates multiple mRNAs: Despite differing donors and endothelial cell types, >50% of iron-induced variability could be explained by the magnitude of transcript downregulation in HHT BOECs with less efficient NMD. The Genotype Tissue Expression (GTEx) Project indicated ND list genes were usually most highly expressed in non-endothelial tissues. However, across 5 major tissues, although 18/486 nonsense and frameshift variants in highly expressed genes were captured in GTEx, none were sufficiently prevalent to obtain genome-wide significant p values for expression quantitative trait loci (GnomAD allele frequencies <0.0005). In conclusion, RNASeq analytics of rare genotype-selected, patient-derived endothelial cells facilitated identification of natural disease-specific and more generic transcriptional signatures. Future studies should evaluate wider relevance and whether injury from external agents is augmented in cells with already high burdens of defective protein production.


title: Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics

abstract: Heterozygous, missense mutations in - or {beta}-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutations impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.


title: Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration

abstract:
Cell migration is a complex process that involves coordinated changes in membrane transport, actin cytoskeleton dynamics, and extracellular matrix remodeling. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a Suppressor of Cytokine Signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 Ubiquitin Ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation, as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading-edge, a process that is required for breast cancer cell migration and invasion.

SUMMARYThe Rab40b/Cul5 complex is an emerging pro-migratory molecular machine. Duncan et al. identify the small GTPase Rap2 as a substrate of the Rab40b/Cul5 complex. They provide evidence that Rab40b/Cul5 ubiquitylates Rap2 to regulate its localization and activity during breast cancer cell migration, ultimately proposing a model by which Rap2 is targeted to the leading-edge plasma membrane to regulate actin dynamics during cell migration.


title: Age-dependent aggregation of ribosomal RNA-binding proteins links deterioration in chromatin stability with loss of proteostasis

abstract: Chromatin instability and loss of protein homeostasis (proteostasis) are two well-established hallmarks of aging, which have been considered largely independent of each other. Using microfluidics and single-cell imaging approaches, we observed that, during the replicative aging of S.cerevisiae, proteostasis decline occurred specifically in the fraction of cells with decreased stability at the ribosomal DNA (rDNA) region. A screen of 170 yeast RNA-binding proteins identified ribosomal RNA (rRNA)- binding proteins as the most enriched group that aggregate upon a decrease in rDNA stability. We further found that loss of rDNA stability contributes to age-dependent aggregation of rRNA-binding proteins through aberrant overproduction of rRNAs. These aggregates negatively impact nucleolar integrity and global proteostasis and hence limit cellular lifespan. Our findings reveal a mechanism underlying the interconnection between chromatin instability and proteostasis decline and highlight the importance of cell-to-cell variability in aging processes.


title: Nucleotide imbalance decouples cell growth from cell proliferation

abstract: Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can accordingly inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is also not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways, causing aberrant biomass production and excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signaling to survive during, and recover from, nucleotide imbalance during S phase. In fact, replication stress signaling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signaling to cope with this metabolic problem, and disrupting the coordination of cell growth and division.


title: Exophers are components of mammalian cell neurobiology in health and disease

abstract: Maintenance of cellular homeostasis is critically important for the survival of cells and organisms. Degradation and recycling of biomolecules and whole organelles is an essential mechanism for maintaining cellular homeostasis. The main systems responsible for these processes are the ubiquitin-proteasome system and autophagy-lysosome pathway. Another mechanism was reported in c. elegans--formation of large, membrane-enclosed projections called "exophers," into which cells direct debris and toxic protein aggregates1. Exophers were shown to act as large, temporary disposal compartments that disconnected from the cells within several hours. Here, we report the discovery of exophers in the mammalian brain, including the brains of humans and mice. Similar to those described in nematodes, the mammalian exophers appear to emanate from the cell body, initially connected by a nanotube, and eventually disconnect. Rare, innate exophers were found in healthy human brain and primary neurons from wild-type mice, presumably mediating transfer of large cargo between cells. The number of exophers increased as an adaptive response under proteostatic stress, e.g., in Alzheimers disease brain or in primary neurons from two tauopathy mouse models, where the exophers likely facilitated expulsion of proteotoxic material. Our findings suggest that exopherogenesis is a rare, innate house-keeping process that is elevated adaptively in response to proteostatic pressure and is a conserved mechanism from nematodes to humans.


title: Rab40c Regulates Focal Adhesions and Protein Phosphatase 6 Activity by Controlling ANKRD28 Ubiquitylation and Degradation

abstract: Rab40c is a SOCS box-containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5 mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesions number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus, leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.


title: Ubiquitination regulates cytoophidium assembly in Schizosaccharomyces pombe

abstract: CTP synthase (CTPS), a metabolic enzyme responsible for the de novo synthesis of CTP, can form filamentous structures termed cytoophidia, which are evolutionarily conserved from bacteria to humans. Here we used Schizosaccharomyces pombe to study the cytoophidium assembly regulation by ubiquitination. We tested the CTP synthases capacity to be epigenetically modified by ubiquitin or be affected by the ubiquitination state of the cell, showed that CTPS is immunoprecipitated with ubiquitin, and that ubiquitination is important for the maintenance of the CTPS filamentous structure in fission yeast. We have identified proteins which are in complex with CTPS, including specific ubiquitination regulators which significantly affect CTPS filamentation, and mapped probable ubiquitination targets on CTPS. Furthermore, we discovered that a cohort of deubiquitinating enzymes is significant for the regulation of cytoophidium morphology. Our study provides a framework for the analysis of the effects that ubiquitination and deubiquitination have on the formation of CTPS filaments.


title: Prdm16 and Notch functionally and physically interact during artery development

abstract: Proper vascular function requires correct arteriovenous (AV) endothelial cell (EC) differentiation. While Notch and its effector Hey2 favor arterial specification, transcription factor (TF) Coup-TFII inhibits Notch activity to induce venous identity. TFs competing with Coup-TFII to orchestrate arterial specification upstream of Notch remain largely unknown. Transcriptional profiling of human and mouse ECs and whole mount in situ hybridization in zebrafish embryos revealed that the TF Prdm16 is exclusively expressed by arterial ECs throughout development, independent of species and hemodynamic factors. Accordingly, prdm16 deficiency in zebrafish perturbed AV specification and caused aberrant shunts between the arterial and venous circulation in an EC- autonomous manner. Prdm16 regulated arterial Notch activity both in vitro and in vivo and simultaneous knockdown of notch aggravated the vascular defects observed in prdm16 morphant zebrafish. In vitro studies revealed that Prdm16 did not alter the levels of the transcriptionally active, intracellular domain of the Notch1 receptor (N1ICD). Rather, it boosted Notch signaling by physically and functionally interacting with both N1ICD and the Notch downstream effector Hey2. Together, these data demonstrate the hitherto unexplored role for Prdm16 during arterial development, by its dual activity on arterial canonical Notch during embryogenesis.

category: developmental biology


title: Tissue-specific inhibition of protein sumoylation uncovers diverse SUMO functions during C. elegans vulval development

abstract: The sumoylation (SUMO) pathway is involved in a variety of processes during C. elegans development, such as gonadal and vulval fate specification, cell cycle progression and maintenance of chromosome structure. The ubiquitous expression of the sumoylation machinery and its involvement in many essential processes has made it difficult to dissect the tissue-specific roles of protein sumoylation and identify the specific target proteins. To overcome these challenges, we have established tools to block protein sumoylation and degrade sumoylated target proteins in a tissue-specific and temporally controlled manner. We employed the auxin-inducible protein degradation system (AID) to down-regulate AID-tagged SUMO E3 ligase GEI-17 or the SUMO ortholog SMO-1, either in the vulval precursor cells (VPCs) or in the gonadal anchor cell (AC). Tissue-specific inhibition of GEI-17 and SMO-1 revealed diverse roles of the SUMO pathway during vulval development, such as AC positioning, basement membrane (BM) breaching, vulval cell fate specification and epithelial morphogenesis. Inhibition of sumoylation in the VPCs resulted in an abnormal shape of the vulval toroids and ectopic cell fusions. Sumoylation of the ETS transcription factor LIN-1 at K169 mediates a subset of these SUMO functions, especially the proper contraction of the ventral vulA toroids. Thus, the SUMO pathway plays diverse roles throughout vulval development.


title: Patterned mechanical feedback establishes a global myosin gradient

abstract: Morphogenesis, the coordinated execution of developmental programs that shape embryos, raises many fundamental questions at the interface between physics and biology. In particular, how the dynamics of active cytoskeletal processes are coordinated across the surface of entire embryos to generate global cell flows is poorly understood. Two distinct regulatory principles have been identified: genetic programs and dynamic response to mechanical stimuli. Despite progress, disentangling these two contributions remains challenging. Here, we combine in toto light sheet microscopy with genetic and optogenetic perturbations of tissue mechanics to examine theoretically predicted dynamic recruitment of non-muscle myosin II to cell junctions during Drosophila embryogenesis. We find dynamic recruitment has a long-range impact on global myosin configuration, and the rate of junction deformation sets the rate of myosin recruitment. Mathematical modeling and high frequency analysis reveal myosin fluctuations on junctions around a mean value set by mechanical feedback. Our model accounts for the early establishment of the global myosin pattern at 80% fidelity. Taken together our results indicate spatially modulated mechanical feedback as a key regulatory input in the establishment of long-range gradients of cytoskeletal configurations and global tissue flow patterns.


title: Subcellular spatial transcriptomics identifies three mechanistically different classes of localizing RNAs

abstract: Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. Here, by subcellular spatial transcriptomics we identify RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and we analyze the mechanisms mediating their localization. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a "default" dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor RNAs (BicD, Bsg25D, hook). Altogether, our study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo.


title: Distributed Feedforward and Feedback Processing across Perisylvian Cortex Supports Human Speech

abstract: Speech production is a complex human function requiring continuous feedforward commands together with reafferent feedback processing. These processes are carried out by distinct frontal and posterior cortical networks, but the degree and timing of their recruitment and dynamics remain unknown. We present a novel deep learning architecture that translates neural signals recorded directly from cortex to an interpretable representational space that can reconstruct speech. We leverage state-of-the-art learnt decoding networks to disentangle feedforward vs. feedback processing. Unlike prevailing models, we find a mixed cortical architecture in which frontal and temporal networks each process both feedforward and feedback information in tandem. We elucidate the timing of feedforward and feedback related processing by quantifying the derived receptive fields. Our approach provides evidence for a surprisingly mixed cortical architecture of speech circuitry together with decoding advances that have important implications for neural prosthetics.


title: The Petasites hybridus CO2-extract (Ze 339) blocks SARS-CoV-2 replication in vitro

abstract: The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2-extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE- and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2 infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 g/mL, repectively. The IC50 values obtained for isopetasin ranged between 0.37-0.88 M for both virus variants, that of remdesivir between 1.53-2.37 M. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-Cov-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.

category: pharmacology and toxicology


title: Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda

abstract: Bovine brucellosis is endemic in Rwanda, although, there is paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n=300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigacion y Tecnologia Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The RBT seroprevalence was 20.7% (62/300), and 2.9% (8/300) with i-ELISA and 2.9% (8/300) using both tests in parallel. Brucella specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n=3), B. abortus (n=3) and B. melitensis (n=5) while Bruce-ladder PCR also identified B. abortus (n=5) and B. melitensis (n=6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures which is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen the national bovine brucellosis control program through vaccination as well as test- and-slaughter.


title: Decrowding Expansion Pathology: Unmasking Previously Invisible Nanostructures and Cells in Intact Human Brain Pathology Specimens

abstract: Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here we present an ExM variant, decrowding expansion pathology (dExPath), which can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and diagnosis.

category: bioengineering


title: Efemp1 modulates elastic fiber formation and mechanics of the extrahepatic bile duct

abstract: EGF-Containing Fibulin Extracellular Matrix Protein 1 (EFEMP1, also called fibulin 3) is an extracellular matrix protein linked in a genome-wide association study to biliary atresia, a fibro-inflammatory disease of the neonatal extrahepatic bile duct. EFEMP1 is expressed in most tissues and Efemp1 null mice have decreased elastic fibers in visceral fascia; however, in contrast to other short fibulins (fibulins 4 and 5), EFEMP1 does not have a role in the development of large elastic fibers, and its overall function remains unclear. We demonstrated that EFEMP1 is expressed in the submucosa of both neonatal and adult mouse and human extrahepatic bile ducts and that, in adult Efemp1+/- mice, elastin organization into fibers is decreased. We used pressure myography, a technique developed to study the mechanics of the vasculature, to show that Efemp1+/- extrahepatic bile ducts are more compliant to luminal pressure, leading to increased circumferential stretch. We conclude that EFEMP1 has an important role in the formation of elastic fibers and mechanical properties of the extrahepatic bile duct. These data suggest that altered expression of EFEMP1 in the extrahepatic bile duct leads to an abnormal response to mechanical stress such as obstruction, potentially explaining the role of EFEMP1 in biliary atresia.

category: physiology


title: Traditional martial arts and shooting training have different effects on auditory fine structure processing ability---Evidence from behavioral tests and fMRI

abstract: Explore the influence of traditional martial arts and shooting training on the ability of auditory temporal fine structure (TFS) processing. Twenty-five college students participated in the experiment, including 8 traditional martial arts practitioners, 8 high-level shooting athletes, and 9 control groups without any regular exercise habits. The BIC (break in interaural correlation) delay threshold and TFS1 test were used to evaluate the temporary storage capacity and sensitivity of TFS, respectively, and a fMRI test was performed after the test. The results found that the traditional martial arts group had stronger TFS sensitivity, while the shooting group had stronger TFS retention ability, and the performance of the behavioral test of the shooting group is related to the fALFF value of the brain area of interest. Traditional martial arts and shooting training have improved the ability of auditory information processing from different angles, diversified exercise habits will lead to the development of diversity in brain structure and function.


title: Disrupted controlling mechanism of salience network on default-mode network and central-executive network in schizophrenia

abstract: Neuroimaging studies suggest that the human brain consists of intrinsically organized large-scale neural networks. Among those networks, the interplay among default-mode network (DMN), salience network (SN), and central-executive network (CEN)has been widely employed to understand the functional interaction patterns in health and diseases. This triple network model suggests that SN causally controls DMN and CEN in healthy individuals. This interaction is often referred to as the dynamic controlling mechanism of SN. However, such interactions are not well understood in individuals with schizophrenia. In this study, we leveraged resting state functional magnetic resonance imaging (fMRI) data of schizophrenia (n = 67) and healthy controls (n = 81) to evaluate the functional interactions among DMN, SN, and CEN using dynamical causal modeling. In healthy controls, our analyses replicated previous findings that SN regulates DMN and CEN activities (Mann-Whitney U test; p < 10-8). In schizophrenia, however, our analyses revealed the disrupted SN-based controlling mechanism on DMN and CEN (Mann-Whitney U test; p < 10-16). These results indicate that the disrupted controlling mechanism of SN on two other neural networks may be a candidate neuroimaging phenotype in schizophrenia.


title: The Effect of Spatial Frequency on the Visual Category Representation in the Macaque Inferior Temporal Cortex

abstract: To expand our knowledge about the object recognition, it is critical to understand the role of spatial frequency (SF) in an object representation that occurs in the inferior temporal (IT) cortex at the final stage of processing the visual information across the ventral visual pathway. Object categories are being recognized hierarchically in at least three levels of abstraction: superordinate (e.g., animal), mid-level (e.g., human face), and subordinate (e.g., face identity). Psychophysical studies have shown rapid access to mid-level category information and low SF (LSF) contents. Although the hierarchical representation of categories has been shown to exist inside the IT cortex, the impact of SF on the multi-level category processing is poorly understood. To gain a deeper understanding of the neural basis of the interaction between SF and category representations at multiple levels, we examined the neural responses within the IT cortex of macaque monkeys viewing several SF-filtered objects. Each stimulus could be either intact or bandpass filtered into either the LSF (coarse shape information) or high SF (HSF) (fine shape information) bands. We found that in both High- and Low-SF contents, the advantage of mid-level representation has not been violated. This evidence suggests that mid-level category boundary maps are strongly represented in the IT cortex and remain unaffected with respect to any changes in the frequency content of stimuli. Our observations indicate the necessity of the HSF content for the superordinate category representation inside the IT cortex. In addition, our findings reveal that the representation of global category information is more dependent on the HSF than the LSF content. Furthermore, the lack of subordinate representation in both LSF and HSF filtered stimuli compared to the intact stimuli provide strong evidence that all SF contents are necessary for fine category visual processing.


title: Vestibular Agnosia is linked to abnormal functional brain networks

abstract: Vestibular Agnosia - where peripheral vestibular activation triggers the usual reflex nystagmus response but with attenuated or no self-motion perception - is found in brain disease with disrupted cortical network functioning, e.g. traumatic brain injury (TBI) or neurodegeneration (Parkinsons Disease). Patients with acute focal hemispheric lesions (e.g. stroke) do not manifest vestibular agnosia. Thus brain network mapping techniques, e.g. resting state functional MRI (rsfMRI), are needed to interrogate functional brain networks mediating vestibular agnosia. Whole-brain rsfMRI was acquired from 39 prospectively recruited acute TBI patients with preserved peripheral vestibular function, along with self-motion perceptual thresholds during passive yaw rotations in the dark. Following quality-control checks, 25 patient scans were analyzed. TBI patients were classified as having vestibular agnosia (n = 11) or not (n = 14) via laboratory testing of self-motion perception. Using independent component analysis, we found altered functional connectivity in the right superior longitudinal fasciculus and left rostral prefrontal cortex in vestibular agnosia. Moreover, regions of interest analyses showed both inter-hemispheric and intra-hemispheric network disruption in vestibular agnosia. In conclusion, our results show that vestibular agnosia is mediated by bilateral anterior and posterior network dysfunction and reveal the distributed brain mechanisms mediating vestibular self-motion perception.


title: The human brain networks mediating the vestibular sensation of self-motion


title: Modulation of behavioral and hippocampal transcriptomic responses in rat prolonged chronic unpredictable stress model by fluoxetine, eicosapentaenoic acid and lipopolysaccharide

abstract: Animal models are widely used to study common stress-induced affective disorders, such as anxiety and depression. Here, we examine behavioral and brain transcriptomic (RNA-seq) responses in rat prolonged chronic unpredictable stress (PCUS) model, and their modulation by 4-week treatment with fluoxetine, eicosapentaenoic acid (EPA), lipopolysaccharide (LPS) and their combinations. Overall, chronic stress produced anxiety-like phenotype, corrected by fluoxetine alone or in combination with EPA or LPS. EPA was anxiolytic in several tests, whereas LPS alone increased anxiety. PCUS evoked pronounced transcriptomic changes in rat hippocampi, differentially expressing >200 genes, while all pharmacological manipulations (except fluoxetine+EPA) affected only few genes. Gpr6, Drd2 and Adora2a were downregulated by chronic stress in a treatment-resistant manner, suggesting highly conserved nature of these pathogenetic genomic responses to chronic stress. Overall, these findings support the validity of rat PCUS paradigm as an effective tool to study stress-related pathologies, and calls for further research to probe how various conventional and novel drugs modulate behavioral and brain transcriptomic biomarkers of chronic stress in rodent models.


title: Changes in natural transformation after salt adaptation

abstract: The exchange of genes between potentially unrelated bacteria is termed horizontal gene transfer (HGT) and is a driving force in bacterial evolution. Natural transformation is one mechanism of HGT where extracellular DNA (eDNA) from the environment is recombined into a host genome. The widespread conservation of transformation in bacterial lineages implies there is a fitness benefit. However, the nature of these benefits and the evolutionary origins of transformation are still unknown. Here, I examine how [~]330 generations or 100 days of serial passage in either constant or increasing salinities impacts the growth rate and transformation efficiency of Pseudomonas stutzeri. While the growth rate generally improved in response to serial transfer, the transformation efficiency of the evolved lineages varied extensively, with only 39-64% of populations undergoing transformation at the end of adaptive evolution. In comparison, 100% of the ancestral populations were able to undergo natural transformation. I also found that evolving P. stutzeri with different cell lysates (or populations of dead cells) minimally affected the growth rate and transformation efficiency, especially in comparison to the pervasiveness with which transformation capacity was lost across the evolved populations. Taken together, I show that the efficiency of eDNA uptake changes over relatively rapid timescales, suggesting that transformation is an adaptive and selectable trait that could be lost in environments where it is not beneficial.