title: Enthalpic and Entropic Contributions to Interleaflet Coupling Drive Domain Registration and Anti-registration in Biological Membrane

abstract: Biological membrane is a complex self-assembly of lipids, sterols and proteins organized as a fluid bilayer of two closely stacked lipid leaflets. Differential molecular interactions among its diverse constituents give rise to heterogeneities in the membrane lateral organization. Under certain conditions, heterogeneities in the two leaflets can be spatially synchronised and exist as registered domains across the bilayer. Several contrasting theories behind mechanisms that induce registration of nanoscale domains have been suggested[1-3]. Following a recent study[4] showing the effect of position of lipid tail unsaturation on domain registration behavior, we decided to develop an analytical theory to elucidate the driving forces that create and maintain domain registry across leaflets. Towards this, we formulated a Hamiltonian for a stacked lattice system where site variables capture the lipid molecular properties such as the position of unsaturation and various other interactions that could drive phase separation and interleaflet coupling. We solve the Hamiltonian using Monte Carlo simulations and create a complete phase diagram that reports the presence or absence of registered domains as a function of various Hamiltonian parameters. We find that the interleaflet coupling should be described as a competing enthalpic contribution due to interaction of lipid tail termini, primarily due to saturated-saturated interactions, and an interleaflet entropic contribution from overlap of unsaturated tail termini. A higher position of unsaturation is seen to provide weaker interleaflet coupling. Thermodynamically stable nanodomains could also be observed for certain points in the parameter space in our bilayer model, which were further verified by carrying out extended Monte Carlo simulations. These persistent non-coalescing registered nanodomains close to the lower end of the accepted nanodomain size range also point towards a possible "nanoscale" emulsion description of lateral heterogeneities in biological membrane leaflets.


title: Pyronaridine Protects Against SARS-CoV-2 in Mouse

abstract:
There are currently relatively few small-molecule antiviral drugs that are either approved or emergency approved for use against SARS-CoV-2. One of these is remdesivir, which was originally repurposed from its use against Ebola and functions by causing early RNA chain termination. We used this as justification to evaluate three molecules we had previously identified computationally with antiviral activity against Ebola and Marburg. Out of these we previously identified pyronaridine, which inhibited the SARS-CoV-2 replication in A549-ACE2 cells. Herein, the in vivo efficacy of pyronaridine has now been assessed in a K18-hACE transgenic mouse model of COVID-19. Pyronaridine treatment demonstrated a statistically significant reduction of viral load in the lungs of SARS CoV-2 infected mice. Furthermore, the pyronaridine treated group reduced lung pathology, which was also associated with significant reduction in the levels of pro-inflammatory cytokines/chemokine and cell infiltration. Notably, pyronaridine inhibited the viral PLpro activity in vitro (IC50 of 1.8 {micro}M) without any effect on Mpro, indicating a possible molecular mechanism involved in its ability to inhibit SARS-CoV-2 replication. Interestingly, pyronaridine also selectively inhibits the host kinase CAMK1 (IC50 of 2.4 {micro}M). We have also generated several pyronaridine analogs to assist in understanding the structure activity relationship for PLpro inhibition. Our results indicate that pyronaridine is a potential therapeutic candidate for COVID-19.

One sentence summaryThere is currently intense interest in discovering small molecules with direct antiviral activity against the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Pyronaridine, an antiviral drug with in vitro activity against Ebola, Marburg and SARS-CoV-2 has now statistically significantly reduced the viral load in mice along with IL-6, TNF-, and IFN-{beta} ultimately demonstrating a protective effect against lung damage by infection to provide a new potential treatment for testing clinically.

category: pharmacology and toxicology


title: Durability of immune responses to the BNT162b2 mRNA vaccine

abstract: The development of the highly efficacious mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology. However, reports of waning vaccine efficacy, coupled with the emergence of variants of concern that are resistant to antibody neutralization, have raised concerns about the potential lack of durability of immunity to vaccination. We recently reported findings from a comprehensive analysis of innate and adaptive immune responses in 56 healthy volunteers who received two doses of the BNT162b2 vaccination. Here, we analyzed antibody responses to the homologous Wu strain as well as several variants of concern, including the emerging Mu (B.1.621) variant, and T cell responses in a subset of these volunteers at six months (day 210 post-primary vaccination) after the second dose. Our data demonstrate a substantial waning of antibody responses and T cell immunity to SARS-CoV-2 and its variants, at 6 months following the second immunization with the BNT162b2 vaccine. Notably, a significant proportion of vaccinees have neutralizing titers below the detection limit, and suggest a 3rd booster immunization might be warranted to enhance the antibody titers and T cell responses.

category: immunology


title: Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice

abstract:
The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination.

We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy.

All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans.

Significance StatementImmunosuppressant regimens are widely used as therapies for a variety of diseases, including autoimmune, inflammatory, and cancer. However, immunosuppressants can impair critical immune responses to vaccination. The impact of standard immunosuppressant use on the critical, developing SARS-CoV-2 vaccination strategies has not been well-described. In this study, we use a mouse model to determine how different immunosuppressant drugs that act through different mechanisms can impair the antibody response to SARS-CoV-2 spike protein, and how modulating these drug regimens may restore antibody levels and function.


title: SARS-CoV-2 Beta variant infection elicits potent lineage-specific and cross-reactive antibodies

abstract:
SARS-CoV-2 Beta variant of concern (VOC) resists neutralization by major classes of antibodies from non-VOC COVID-19 patients and vaccinated individuals. Here, serum of Beta variant infected patients revealed reduced cross-neutralization of non-VOC virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of novel VOC-specific clonotypes and accommodation of VOC-defining amino acids into a major non-VOC antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with non-VOC-elicited antibodies, including a public VH1-58 clonotype targeting the RBD ridge independent of VOC mutations. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift with implications for design of next-generation vaccines and therapeutics.

One sentence summarySARS-CoV-2 Beta variant elicits lineage-specific antibodies and antibodies with neutralizing breadth against wild-type virus and VOCs.


title: Ad26.COV2.S Prevents SARS-CoV-2 Induced Pathways of Inflammation and Thrombosis in Hamsters

abstract:
Syrian golden hamsters exhibit features of severe disease after SARS-CoV-2 challenge and are therefore useful models of COVID-19 pathogenesis and prevention with vaccines. Recent studies have shown that SARS-CoV-2 infection stimulates type I interferon, myeloid, and inflammatory signatures similar to human disease, and that weight loss can be prevented with vaccines. However, the impact of vaccination on transcriptional programs associated with COVID-19 pathogenesis and protective adaptive immune responses is unknown. Here we show that SARS-CoV-2 challenge in hamsters stimulates antiviral, myeloid, and inflammatory programs as well as signatures of complement and thrombosis associated with human COVID-19. Notably, single dose immunization with Ad26.COV2.S, an adenovirus serotype 26 vector (Ad26)-based vaccine expressing a stabilized SARS-CoV-2 spike protein, prevents the upregulation of these pathways such that the gene expression profiles of vaccinated hamsters are comparable to uninfected animals. Finally, we show that Ad26.COV2.S vaccination induces T and B cell signatures that correlate with binding and neutralizing antibody responses. These data provide further insights into the mechanisms of Ad26.COV2.S based protection against severe COVID-19 in hamsters.

Author SummaryIn this study, we show that vaccination with Ad26.COV2.S protected SARS-CoV-2 challenged hamsters from developing severe COVID-19 disease by attenuating excessive proinflammatory responses, such as IL-6 and IL-1, macrophages and neutrophils signaling. Ad26 vaccination also prevented the upregulation of pathways associated with thrombosis such coagulation and clotting cascades associated with infection, and the transcriptomic profiles of vaccinated animals were largely comparable to control uninfected hamsters. In contrast, SARS-CoV-2 challenged unvaccinated hamsters showed significant increase of these proinflammatory and prothrombotic pathways and significant weight loss compared to vaccinated hamsters.

category: microbiology


title: Bile Acid Regulates the Colonization and Dissemination of Candida albicans from the Gastrointestinal Tract by Controlling Host Defense System and Microbiota

abstract:
Candida albicans (CA), a commensal and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract and causes life-threatening infections. Antibiotic-induced gut dysbiosis is a major risk factor for increased CA colonization and dissemination from the GI tract. We identified a significant increase of taurocholic acid (TCA), a major bile acid in antibiotic-treated mice susceptible to CA infection. In vivo findings indicate that administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in wild type and immunosuppressed mice. Treatment with TCA significantly reduced mRNA expression of immune genes ang4 and Cxcr3 in the colon. In addition, TCA significantly decreased the relative abundance of three culturable species of commensal bacteria, Turicibacter sanguinis, Lactobacillus johnsonii, and Clostridium celatum, in both cecal contents and mucosal scrapings from colon. Taken together, our results indicate that TCA promotes fungal colonization and dissemination of CA from the GI tract by controlling host defense system and intestinal microbiota that play a critical role in regulating CA in the intestine.

ImportanceBroad-spectrum antibiotics, FDA-approved bile acid drugs, and probiotics used to control metabolic and infectious diseases profoundly alter the level of TCA in the gut. Furthermore, TCA level is highly altered in a subset of cancer, colitis and surgery patients who are highly susceptible to CA infection. Inadvertently, these therapies and disease conditions could be either promoting CA colonization and dissemination. Our findings indicate that TCA alone can induce fungal colonization and dissemination from the intestine. Results from this study will have a significant impact in understanding how bile acids interact with the microbiota and host in regulating invasive fungal infections that originate from the intestine and to develop potential new antifungal therapeutics.


title: Biodegradation of naphthalene mediated by the plant growth promoting rhizobacteria

abstract:
We compared the ability of two bacterial strains, Paenibacillus polymyxa A26 and P. polymyxa A26Sfp, for biodegradation of naphthalene (NAP). The studies were performed under simulated laboratory conditions, in liquid medium and soil with different carbon sources, pH and salt contents. Changes in the luminescence inhibition of Aliivibrio fischeri, as an indicator of the baseline toxicity, were observed in degradation mixtures during 7 days of incubation. While both strains expressed the best growth and NAP degradation ability in the minimal salt medium containing sucrose and 5% NaCl at pH 8, the mutant strain remained effective even under extreme conditions.

A26Sfp was found to be an efficient and potentially industrially important polycyclic aromatic hydrocarbon degradation strain. Its extracellular polysaccharide production is 30% and glucan production twice that of the wild type A 26. The surface tension reduction ability was ascertained as 25-30% increased emulsification ability.


title: A model screening pipeline for bile acid converting anti-Clostridioides difficile bacteria reveals unique biotherapeutic potential of Peptacetobacter hiranonis

abstract: Clostridioides difficile is an antibiotic-resistant bacterium that causes serious, toxin-mediated enteric disease in humans and animals. Gut dysbiosis and resultant alterations in the intestinal bile acid profile play an important role in the pathogenesis of C. difficile infection (CDI). Restoration of the gut microbiota and re-establishment of bacterial bile acid metabolism using fecal microbiota transplantation (FMT) has been established as a promising strategy against this disease, although this method has several limitations. Thus, a more defined and precise microbiota-based approach using bacteria that biotransform primary bile acids into secondary bile acids could effectively overcome these limitations and control CDI. Therefore, a screening pipeline was developed to isolate bile acid converting bacteria from fecal samples. Dogs were selected as a model CDI-resistant microbiota donor for this pipeline, which yielded a novel Peptacetobacter hiranonis strain that possesses unique anti-C. difficile properties, and both bile acid deconjugation and 7- dehydroxylating activities to perform bile acid conversion. The screening pipeline included a set of in vitro tests along with a precision in vivo gut colonization and bile acid conversion test using altered Schadler flora (ASF) colonized mice. In addition, this pipeline also provided essential information on the growth requirements for screening and cultivating the candidate bacterium, its survival in a CDI predisposing environment, and potential pathogenicity. The model pipeline documented here yielded multiple bile acid converting bacteria, including a P. hiranonis isolate with unique anti-C. difficile biotherapeutic potential, which can be further tested in subsequent preclinical and human clinical trials.


title: The unforeseen intracellular lifestyle of Enterococcus faecalis in hepatocytes

abstract: Enterococcus faecalis is a bacterial species present at a sub-dominant level in the human gut microbiota. This commensal turns into an opportunistic pathogen under specific conditions involving dysbiosis and host immune deficiency. E. faecalis is also the only intestinal pathobiont identified to date as contributing to liver damage in alcoholic liver disease. We have previously observed that E. faecalis is internalized in hepatocytes. Here, the survival and fate of E. faecalis was examined in hepatocytes, the main epithelial cell type in the liver. Although referred to as an extracellular pathogen, we demonstrate that E. faecalis is able to survive and divide in hepatocytes, and form intracellular clusters in two distinct hepatocyte cell lines, in primary mouse hepatocytes, as well as in vivo. This novel process extends to kidney cells. Unravelling the intracellular lifestyle of E. faecalis, our findings contribute to the understanding of pathobiont-driven diseases.


title: Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics

abstract: Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug-microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at -80{degrees}C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.


title: Drivers and Determinants of Strain Dynamics Following Faecal Microbiota Transplantation

abstract: Faecal microbiota transplantation (FMT) is an efficacious therapeutic intervention, but its clinical mode of action and underlying microbiome dynamics remain poorly understood. Here, we analysed the metagenomes associated with 142 FMTs, in a time series-based meta-study across five disease indications. We quantified strain-level dynamics of 1,089 microbial species based on their pangenome, complemented with 47,548 newly constructed metagenome-assembled genomes. Using subsets of procedural-, host- and microbiome-based variables, LASSO-regularised regression models accurately predicted the colonisation and resilience of donor and recipient microbes, as well as turnover of individual species. Linking this to putative ecological mechanisms, we found these sets of variables to be informative of the underlying processes that shape the post-FMT gut microbiome. Recipient factors and complementarity of donor and recipient microbiomes, encompassing entire communities to individual strains, were the main determinants of individual strain population dynamics, and mostly independent of clinical outcomes. Recipient community state and the degree of residual strain depletion provided a neutral baseline for donor strain colonisation success, in addition to inhibitive priority effects between species and conspecific strains, as well as putatively adaptive processes. Our results suggest promising tunable parameters to enhance donor flora colonisation or recipient flora displacement in clinical practice, towards the development of more targeted and personalised therapies.


title: Molecular characterization of Mycobacterium ulcerans DNA gyrase and identification of mutations reduced susceptibility against quinolones in vitro

abstract: Buruli ulcer disease is a neglected necrotizing and disabling cutaneous tropical illness caused by Mycobacterium ulcerans (Mul). Fluoroquinolone (FQ), used in the treatment of this disease, has been known to act by inhibiting the enzymatic activities of DNA gyrase; however, the detailed molecular basis of these characteristics and the FQ resistance mechanisms in Mul remains unknown. This study investigated the detailed molecular mechanism of Mul DNA gyrase and the contribution of FQ resistance in vitro using recombinant proteins from the Mul subsp. shinshuense and Agy99 strains with reduced sensitivity to FQs. The IC50 of FQs against Ala91Vla and Asp95Gly mutants of Mul shinshuense and Agy99 GyrA subunits were 3.7- to 42.0-fold higher than those against wild-type enzyme. Similarly, the CC25 was 10- to 210-fold higher than those for the WT enzyme. Furthermore, the interaction between the amino acid residues of WT/mutant Mul DNA gyrase and FQ side chains was assessed via molecular docking studies. This is the first detailed study showing the contribution of Mul DNA GyrA subunit mutations to reduce the susceptibility against FQs.

category: molecular biology


title: Prime Editing Efficiency and Fidelity are Enhanced in the Absence of Mismatch Repair

abstract: Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, the efficiency of PE varies widely and depends not only on the genomic region targeted, but also on the genetic background of the edited cell. To determine which cellular factors affect PE efficiency, we carried out a focused genetic screen targeting 32 DNA repair factors, spanning all reported repair pathways. We show that, depending on cell line and type of edit, ablation of mismatch repair (MMR) affords a 2-17 fold increase in PE efficiency, across several human cell lines, types of edits and genomic loci. The accumulation of the key MMR factors MLH1 and MSH2 at PE sites argues for direct involvement of MMR in PE control. Our results shed new light on the mechanism of PE and suggest how its efficiency might be optimised.


title: Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome.

abstract: Leishmania are unicellular parasites that cause human and animal disease. Alongside other organisms in kinetoplastida, they have evolved an unusual genome architecture that requires all RNA polymerase II transcribed genes to be expressed constitutively, with transcriptional start regions denoted by histone variants and histone lysine acetylation. However, the way these chromatin marks are interpreted by the cell is not understood. Seven predicted bromodomain factors (BDF1-7), the reader modules for acetyl-lysine, were identified across Leishmania genomes. Using L. mexicana as a model, Cas9-driven gene deletions indicate that BDF1-5 are essential for promastigote survival, whilst DiCre inducible gene deletion of the dual bromodomain factor BDF5 identified it to be essential for both promastigotes and amastigotes. ChIP-seq assessment of BDF5s genomic distribution revealed it as highly enriched at transcriptional start sites. Using an optimised proximity proteomic and phosphoproteomic technique, XL-BioID, we defined the BDF5-proximal environment to be enriched for other bromodomain factors, histone acetyltransferase 2, and proteins essential for transcriptional activity and RNA processing. Inducible deletion of BDF5, led to a disruption of pol II transcriptional activity and global defects in gene expression. Our results indicate the requirement of Leishmania to interpret histone acetylation marks for normal levels of gene expression and thus cellular viability.

category: cell biology


title: Large-scale comparative genomics of Salmonella enterica to refine the organization of the global Salmonella population structure

abstract:
1.Since the introduction of the White-Kauffmann-Le Minor (WKL) scheme for Salmonella serotyping, the nomenclature remains the most widely used for reporting the disease prevalence of Salmonella enterica across the globe. With the advent of whole genome sequencing (WGS), traditional serotyping has been increasingly replaced by in-silico methods that couple the detection of genetic variations in antigenic determinants with sequence-based typing. However, despite the integration of genomic-based typing by in-silico serotyping tools such as SeqSero2 and SISTR, in-silico serotyping in certain contexts remains ambiguous and insufficiently informative due to polyphyletic serovars. Furthermore, in spite of the widespread acknowledgement of polyphyly from genomic studies, the serotyping nomenclature remains unaltered. To prompt refinements to the Salmonella typing nomenclature for disease reporting, we herein performed a systematic characterization of putative polyphyletic serovars and the global Salmonella population structure by comparing 180,098 Salmonella genomes (representing 723 predicted serovars) from GenomeTrakr and PubMLST databases. We identified a range of core genome MLST typing thresholds that result in stable population structure, potentially suitable as the foundation of a genomic-based typing nomenclature for longitudinal surveillance. From the genomic comparisons of hundreds of predicted serovars, we demonstrated that in-silico serotyping classifications do not consistently reflect the population divergence observed at the genomic level. The organization of Salmonella subpopulations based on antigenic determinants can be confounded by homologous recombination and niche adaptation, resulting in shared classification of highly divergent genomes and misleading distinction between highly similar genomes. In consideration of the pivotal role of Salmonella serotyping, a compendium of putative polyphyletic serovars was compiled and made publicly available to provide additional context for future interpretations of in-silico serotyping results in disease surveillance settings. To refine the typing nomenclatures used in Salmonella surveillance reports, we foresee an improved typing scheme to be a hybrid that integrates both genomic and antigenic information such that the resolution from WGS is leveraged to improve the precision of subpopulation classifications while preserving the common names defined by the WKL scheme. Lastly, we stress the importance of controlled vocabulary integration for typing information in open data settings in order for the global Salmonella population dynamics to be fully trackable.

2. Impact StatementSalmonella enterica (S. enterica) is a major foodborne pathogen responsible for an annual incidence rate of more than 90 million cases of foodborne illnesses worldwide. To surveil the high order Salmonella lineages, compare disease prevalence across jurisdictions worldwide, and inform risk assessments, in-silico serotyping has been established as the gold standard for typing the bacteria. However, despite previous Salmonella genomic studies reporting discordance between phylogenomic clades and serovars, refinements have yet been made to the serotyping scheme. Here, we analyzed over 180,000 Salmonella genomes representing 723 predicted serovars to subdivide the population into evolutionarily stable clusters in order to propose a stable organization of the Salmonella population structure that can form the basis of a genomic-based typing scheme for the pathogen. We described numerous instances in which genomes between serotypes are more similar than genomes within a serotype to reflect the inconsistencies of subpopulation classifications based on antigenic determinants. Moreover, we found inconsistencies between predicted serovars and reported serovars which highlighted potential errors in existing in-silico serotyping tools and the need to implement controlled vocabularies for reporting Salmonella subtypes in public databases. The findings of our study aim to motivate the future development of a standardized genomic-based typing nomenclature that more accurately captures the natural populations of S. enterica.

3. Data SummaryThe assembly accession numbers of the genomes analyzed in this study (n = 204,952) and the associated metadata (e.g. sampling location, collection date, FTP address for retrieval) are documented in Table S1. The GenomeTrakr genomes were retrieved from the National Center for Biological Information GenBank database. The PubMLST genomes were retrieved using the BIGSdb API.

category: genomics


title: Structures of core eukaryotic protein complexes

abstract:
Protein-protein interactions play critical roles in biology, but despite decades of effort, the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions that have not yet been identified. Here, we take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes, as represented within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of S. cerevisiae proteins and build models for strongly predicted protein assemblies with two to five components. Comparison to existing interaction and structural data suggests that these predictions are likely to be quite accurate. We provide structure models spanning almost all key processes in Eukaryotic cells for 104 protein assemblies which have not been previously identified, and 608 which have not been structurally characterized.

One-sentence summaryWe take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes.

category: biochemistry


title: Light chain 2 is a Tctex-type related axonemal dynein light chain that regulates directional ciliary motility in Trypanosoma brucei

abstract:
Flagellar motility is essential for the cell morphology, viability, and virulence of pathogenic kinetoplastids, including trypanosomes. Trypanosoma brucei flagella exhibit a bending wave that propagates from the flagellums tip to its base, rather than base-to-tip as in other eukaryotes. Thousands of dynein motor proteins coordinate their activity to drive ciliary bending wave propagation. Dynein- associated light and intermediate chains regulate the biophysical mechanisms of axonemal dynein. Tctex- type outer arm dynein light chain 2 (LC2) regulates flagellar bending wave propagation direction, amplitude, and frequency in Chlamydomonas reinhardtii. However, the role of Tctex-type light chains in regulating T. brucei motility is unknown. Here, we used a combination of bioinformatics, in-situ molecular tagging, and immunofluorescence microscopy to identify a Tctex-type light chain in the procyclic form of T. brucei (TbLC2). We knocked down TbLC2 expression using RNAi, rescued the knockdown with eGFP- tagged TbLC2, and quantified TbLC2s effects on trypanosome cell biology and biophysics. We found that TbLC2 knockdown resulted in kinetoplast mislocalization and the formation of multiple cell clusters in cell culture. We also found that TbLC2 knockdown reduced the directional persistence of trypanosome cell swimming, induced an asymmetric ciliary bending waveform, modulated the bias between the base-to- tip and tip-to-base beating modes, and increased the beating frequency. Together, our findings are consistent with a model of TbLC2 as a down-regulator of axonemal dynein activity that stabilizes the forward tip-to-base beating ciliary waveform characteristic of trypanosome cells. Our work sheds light on axonemal dynein regulation mechanisms that contribute to pathogenic kinetoplastids unique tip-to-base ciliary beating nature and how those mechanisms underlie dynein-driven ciliary motility more generally.

Author SummaryKinetoplastea is a class of ciliated protists that include parasitic trypanosomes, which cause severe disease in people and livestock in tropical regions across the globe. All trypanosomes, including Trypanosoma brucei, require a cilium to provide propulsive force for directional swimming motility, host immune evasion, and various aspects of their cell cycle. Thus, a functional cilium is essential for the virulence of the parasite.

Trypanosome cilia exhibit a unique tip-to-base beating mechanism, different from the base-to-tip beating of most other eukaryotic cilia. Multiple ciliary proteins are involved in the complex biophysical and biochemical mechanisms that underly the trypanosome ciliary beating. These include dynein motor proteins that power the beat, dynein-related light chains that regulate the beat, and many other proteins in the nexin-dynein regulatory complex, in the radial spokes, and associated with the central pair of microtubules, for example.

Here, we identify a Tctex-type dynein light chain in T. brucei that we named TbLC2 because it has sequence homology, structural similarity, and ciliary localization like LC2 homologs in other organisms. We demonstrate that TbLC2 has critical dynein regulatory functions, with implications on the unique aspects of trypanosome ciliary beating and cellular swimming motility. Our study represents an additional step toward understanding the functions of the trypanosome ciliary proteome, which could provide novel therapeutic targets against the unique aspects of trypanosome ciliary motility.

category: biophysics


title: Hypoxia-directed tumor targeting of CRISPR/Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

abstract: Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia responsive element (HRE) sequences within the promoter of target genes. HRE driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene and the CRISPR/Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the CMV and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions, while maintaining tight regulation with no significant changes to cell viability in normoxia.

category: cancer biology


title: Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

abstract: Genome-wide functional genetic screens have been successful in discovering genotype-phenotype relationships and in engineering new phenotypes. While broadly applied in mammalian cell lines and in E. coli, use in non-conventional microorganisms has been limited, in part, due to the inability to accurately design high activity CRISPR guides in such species. Here, we develop an experimental-computational approach to sgRNA design that is specific to an organism of choice, in this case the oleaginous yeast Yarrowia lipolytica. A negative selection screen in the absence of non-homologous end-joining, the dominant DNA repair mechanism, was used to generate single guide RNA (sgRNA) activity profiles for both SpCas9 and LbCas12a. This genome-wide data served as input to a deep learning algorithm, DeepGuide, that is able to accurately predict guide activity. DeepGuide uses unsupervised learning to obtain a compressed representation of the genome, followed by supervised learning to map sgRNA sequence, genomic context, and epigenetic features with guide activity. Experimental validation, both genome-wide and with a subset of selected genes, confirms DeepGuides ability to accurately predict high activity sgRNAs. DeepGuide provides an organism specific predictor of CRISPR guide activity that could be broadly applied to fungal species, prokaryotes, and other non-conventional organisms.

category: synthetic biology


title: Transition of Vibrio cholerae through a natural host induces resistance to environmental changes

abstract: The pandemic-related strains of Vibrio cholerae are known to cause diarrheal disease in animal hosts. These bacteria must overcome rapid changes in their environment, such as the transition from fresh water to the gastrointestinal system of their host. To study the morphological adjustments during environmental transitions, we used zebrafish as a natural host. Using a combination of fluorescent light microscopy, cryogenic electron tomography and serial block face scanning electron microscopy, we studied the structural changes that occur during the infection cycle. We show that the transition from an artificial nutrient-rich environment to a nutrient-poor environment has a dramatic impact on the cell shape, most notably membrane dehiscence. In contrast, excreted bacteria from the host retain a uniform distance between the membranes as well as their vibrioid shape. Inside the intestine, V. cholerae cells predominantly colonized the anterior to mid-gut, forming micro-colonies associated with the microvilli as well as within the lumen. The cells retained their vibrioid shape but changed their cell-length depending on their localization. Our results demonstrate dynamic changes in morphological characteristics of V. cholerae during the transition between the different environments, and we propose that these structural changes are critical for the pathogens ability to colonize host tissues.


title: Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health

abstract:
The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.

Author SummaryThe intestinal microbiota is an important determinant of health and disease and is shaped by the environment of the gut lumen. The nervous system of the intestine, the enteric nervous system (ENS), helps maintain many aspects of intestinal health including a healthy microbiota. We used zebrafish with a genetic mutation that impedes ENS formation to investigate how the ENS prevents pathogenic shifts in the microbiota. We found that mutants lacking an ENS have a lower luminal pH, higher load of pathogenic bacteria, and intestinal inflammation. We showed that correcting the low pH, using the commonly prescribed pharmacological agent omeprazole, restored the microbiota and prevented intestinal inflammation. Conversely, we found that lowering the luminal pH of wild type animals, using the drug acetazolamide, caused expansion of pathogenic bacteria and increased intestinal inflammation. From these experiments, we conclude that a primary function of the ENS is to maintain normal luminal pH, thereby constraining intestinal microbiota community composition and promoting intestinal health.


title: Efficient single copy integration via homology-directed repair (scHDR) by 5'modification of large DNA donor fragments in mice

abstract: CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5biotinylation. Thus, we suggest that a combination of a 5biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.


title: Transmembrane Signaling Regulates the Remodeling of the Actin Cytoskeleton:Roles of PKC and Adducin

abstract: We tested the hypothesis that Pkc53E regulates adducin to orchestrate the remodeling of the membrane skeleton following the transmembrane GPCR-Gq signaling. Adducin is a known substrate of PKC and is critical for the assembly of the membrane skeleton by cross-linking actin filaments with the spectrin network. In Drosophila photoreceptors, loss of function in pkc53E leads to retinal degeneration while Pkc53E-RNAi negatively impacts the actin cytoskeleton of the visual organelle rhabdomeres. Unexpectedly, Pkc53E-RNAi enhances the degeneration caused by the loss of PLC{beta}4 (norpAP24). We show that when PLC{beta}4 is absent Plc21C may be activated instead for activating Pkc53E. We investigate whether Pkc53E phosphorylates adducin in vivo and observed that levels of phosphorylated adducin at the conserved PKC site were greatly reduced in a null allele of pkc53E. We show Pkc53E-RNAi did not modify adducin-RNAi, which exerts a more severe effect on the actin cytoskeleton. Moreover, overexpression of the mCherry-tagged adducin that appears to act in a dominant-negative manner interferes with the spectrin interaction leading to the apical expansion of rhabdomeres similar to that of {beta}-spectrin-RNAi. We performed epistasis analysis and show that double mutants of the tagged adducin and Pkc53E-RNAi display the expansion phenotype at the eclosion, but progress to severe degeneration in adult photoreceptors. Together, most of our findings support that adducin is likely regulated by Pkc53E in Drosophila photoreceptors.


title: The YAP/TAZ antagonist, PTPN14, stabilizes SMAD4 through direct interactions in endothelial cells: Implications for Hereditary Hemorrhagic Telangiectasia

abstract:
Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular condition caused by germline heterozygous loss-of-function mutations of ENG, AVCRL1, and occasionally SMAD4, encoding components of TGF{beta}/BMP signaling. Telangiectases occur in most HHT patients, and pulmonary, visceral, or cerebral arteriovenous malformations (AVMs) occur in 20-50%, but our understanding of how HHT mutations disrupt down-stream signaling pathways causing clinical manifestations, and why some patients suffer more serious sequelae, is incomplete. We previously showed that genetic variation within PTPN14 at rs2936018 associates with presentation of PAVM in HHT patients. Here we show rs2936018 is a cis-eQTL for PTPN14, with lower expression of the HHT at-risk allele, and that in primary human endothelial cells, PTPN14 physically interacts with the transcription factor, SMAD4, protecting it from ubiquitylation to support higher SMAD4 expression levels.

In a panel of 69 lung samples, we find Ptpn14 RNA expression correlates with markers of angiogenesis, lymphangiogenesis, cell-cell interaction, BMP signaling and rho kinase signaling, indicating preferential expression in endothelial cells. RNAScope in situ hybridization analysis and use of transgenic Ptpn14-reporter mice (Ptpn14.tm1(KOMP) Vlcg) show that Ptpn14 is predominantly but not exclusively expressed in lymphatic and vascular ECs of lung, heart and skin. In lung, Ptpn14, Acvrl1, Eng and SMAD4 expression are tightly correlated as well as with Flt1, Ece1, Sash1, and Mapk3k. Additionally, Ptpn14, Acvrl1, Eng and SMAD4 show strong expression correlation with components of G protein coupled receptor (GPCR) signaling pathways impacting rho kinase, Cdc42, a regulator of cell migration. We report, for the first time, that in primary human endothelial cells PTPN14 binds SMAD4, as demonstrated by co-immunoprecipitation studies and confirmed by proximity ligation assay. In the nucleus, PTPN14 stabilizes SMAD4, protecting it from ubiquitylation and turnover and potentiating basal transcriptional activity from BMP and TGF{beta} responsive reporters, whereas in the cytoplasm PTPN14 sequesters phospho-TAZ (pTAZ) leading to TAZ turnover. PTPN14 therefore provides a physical link supporting BMP/ALK-1/SMAD4 signaling while inhibiting YAP/TAZ signaling in ECs to regulate a balance between these two pathways to maintain vascular stability. Polymorphisms in PTPN14 may alter tonic BMP/ALK-1/SMAD4 and TGF{beta}/TGF{beta}RI/SMAD4 signaling to magnify ligand-mediated responses. In this way, genetic variation within PTPN14 may influence clinical outcomes of HHT through its action on SMAD4.


title: Extracellular matrix stiffness and TGFβ2 regulate YAP/TAZ activity in human trabecular meshwork cells

abstract: Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGF{beta}2) levels in aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM (GTM) cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGF{beta}2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ transcriptional activity potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGF{beta}2 increased YAP/TAZ nuclear localization in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGF{beta}2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGF{beta}2-induced HTM/GTM hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.


title: Structural Insights into the BRAF Monomer-to-dimer Transition Mediated by RAS Binding

abstract: RAF kinases are essential effectors of RAS, but how RAS binding initiates the conformational changes needed for autoinhibited RAF monomers to form active dimers has remained unclear. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 [A] resolution, respectively. In both autoinhibited, monomeric structures, the RAS binding domain (RBD) of BRAF is resolved, revealing that the RBD forms an extensive contact interface with the 14-3-3 protomer bound to the BRAF C-terminalsite and that key basic residues required for RBD-RAS binding are exposed. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the center of the RBD:14-3-3 interface have a dual function, first contributing to RAF autoinhibition and then to the full spectrum of RAS-RBD interactions.


title: Structure and function of N-acetylglucosamine kinase illuminates the catalytic mechanism of ROK kinases

abstract: N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell walls. Many organisms recycle GlcNAc from the cell wall or metabolise environmental GlcNAc. The first step in GlcNAc metabolism is phosphorylation to GlcNAc-6-phosphate. In bacteria, the ROK family kinase NagK performs this activity. Although ROK kinases have been studied extensively, no ternary complex showing the two substrates has yet been observed. Here, we solved the structure of NagK from the human pathogen Plesiomonas shigelloides in complex with GlcNAc and the ATP analogue AMP-PNP. Surprisingly, PsNagK showed two conformational changes associated with the binding of each substrate. Consistent with this, the enzyme showed a sequential random enzyme mechanism. This indicates that the enzyme acts as a coordinated unit responding to each interaction. Molecular dynamics modelling of catalytic ion binding confirmed the location of the essential catalytic metal. Site-directed mutagenesis confirmed the catalytic base, and that the metal coordinating residue is essential. Together, this study provides the most comprehensive insight into the activity of a ROK kinase.


title: A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction

abstract: CRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this gene-editing technique is quite accurate in the target region, there may be many unplanned off-target edited sites. Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of off-target sites) produced by experimental techniques to detect off-target sites with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect off-target sites, was used to produce a dataset of unprecedented scale and quality (more than 200,000 off-target sites over 110 guide RNAs). In addition, the same study included GUIDE-seq experiments for 58 of the guide RNAs to produce in vivo measurements of off-target sites. Here, we fill the gap in previous computational methods by utilizing these data to perform a systematic evaluation of data processing and formulation of the CRISPR off-target site prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive off-target sites to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between the guide RNA and the off-target site as a feature. Finally, we present predictive off-target in vivo models based on transfer learning from in vitro. Our conclusions will be instrumental to any future development of an off-target predictor based on high-throughput datasets.

category: bioinformatics


title: Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

abstract: Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1s lumenal domain. Phosphorylation of IRE1s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans- autophosphorylation, which in turn drives IRE1s RNase activity.


title: A gut commensal niche regulates stable association of a multispecies microbiota

abstract:
The intestines of animals are typically colonized by a complex, relatively stable microbiota that influences health and fitness, but the underlying mechanisms of colonization remain poorly understood. As a typical animal, the fruit fly, Drosophila melanogaster, is associated with a consistent set of commensal bacterial species, yet the reason for this consistency is unknown. Here, we use gnotobiotic flies, microscopy, and microbial pulse-chase protocols to show that a commensal niche exists within the proventriculus region of the Drosophila foregut that selectively binds bacteria with exquisite strain-level specificity. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus physically remodels the niche to favor secondary colonization by Acetobacter. Our results provide a mechanistic framework for understanding the establishment and stability of an intestinal microbiome.

One-Sentence SummaryA strain-specific set of bacteria inhabits a defined spatial region of the Drosophila gut that forms a commensal niche.


title: Variation in the response to malaria in Diversity Outbred mice

abstract: We infected Diversity Outbred mice with Plasmodium chabaudi to better understand how the host response to infection can vary and to try to identify genetic loci responsible for this variation. We identified two loci correlating with binary traits: one on chromosome two was linked to undetectable parasite loads and another on chromosome ten which was linked to death. Though we tested many variable traits, none of those reached statistical significance using the 489 mice we tested.


title: Association between Cognitive Function and Large Optic Nerve Cupping, Accounting for Cup-Disc-Ratio Genetic Risk Score

abstract:
PurposeTo investigate if accounting for a cup-to-disc ratio (CDR) genetic risk score (GRS) modified the association between large CDR and cognitive function among women.

DesignThis was a retrospective study using data from the Womens Health Initiative.

MethodsPatients with glaucoma or ocular hypertension were excluded. Large CDR was defined as [≥] 0.6 in either eye. Cognitive function was measured by the Modified Mini-Mental State Examination (3MSE). We used the combined effects from 13 single nucleotide polymorphisms (SNPs) to formulate the GRS for CDR. We used logistic regression to investigate associations between weighted GRS and large CDR, then a linear regression to assess the association between weighted GRS and 3MSE scores, and between weighted GRS, CDR, and 3MSE scores, adjusted for demographic and clinical characteristics.

ResultsFinal analyses included 1,196 White women with mean age of 69.60 {+/-} 3.62 years and 7.27% with large CDR. Mean GRS in women with and without large CDR was 1.51 {+/-} 0.31 vs. 1.41 {+/-} 0.36, respectively (p = 0.004). The odds of large CDR for a one unit increase in GRS was 2.30 (95% CI: (1.22, 4.36), p = 0.011). Adding the CDR GRS in the model with CDR and 3MSE, women with large CDR still had statistically significantly lower 3MSE scores than those without large CDR, yielding a predicted mean difference in 3MSE scores of 0.84 (p = 0.007).

ConclusionsIndependent of the CDR GRS, women with large CDR had a lower cognitive function.

category: genetics


title: Crystal Structures of Ferritin Grown by the Microbatch Method in the Presence of Agarose and Electric Field Shows Enhanced Metal Binding

abstract: Ferritin is an ubiquitous iron storage protein found in all kingdoms of life. Ferritin is essential for iron homeostasis and is involved in a wide range of physiological and pathological processes. Several structures of ferritin in complex with small molecules and metal ions have been reported. Here we report the crystal structures of Horse Spleen Ferritin, in which the crystals were grown by employing a novel approach adopting the microbatch experiments performed in the presence and absence of electric field using a 2% agarose pellet of CdSO4. We observed that 1) these structures contain increased number of Cd ions as compared to the crystallization of same protein by others using different methods. 2) The externally applied electric field reduced the number of nucleation and with fewer nucleation the size of the crystals increased. 3) There is no significant conformational change observed among these structures. 4) Irrespective of the externally applied electric field, this agarose microbatch crystallization method facilitates the retaining of increased number of bound metal ions with ferritin to mimic the possible in vivo environment.


title: The parallel-stranded d(CGA) duplex is a highly predictable structural motif with two conformationally distinct strands

abstract: DNA can adopt non-canonical structures that have important biological functions while also providing structural diversity for nanotechnology applications. Here, we describe the crystal structures of two oligonucleotides composed of d(CGA) triplet repeats in the parallel-stranded duplex form. The structure determination of four unique d(CGA)-based parallel-stranded duplexes across two crystal structures has allowed us to characterize and establish structural parameters of d(CGA) triplets in the parallel-stranded duplex form. Our results show that d(CGA) units are highly uniform, but that each strand in the duplex is structurally unique and has a distinct role in accommodating structural asymmetries induced by the C-CH+ base pair.


title: Are Machines-learning Methods More Efficient than Humans in Triaging Literature for Systematic Reviews?

abstract: Systematic literature reviews provide rigorous assessments of clinical, cost-effectiveness, and humanistic data. Accordingly, there is a growing trend worldwide among healthcare agencies and decision-makers to require them in order to make informed decisions. Because these reviews are labor-intensive and time consuming, we applied advanced analytic methods (AAM) to determine if machine learning methods could classify abstracts as well as humans. Literature searches were run for metastatic non-small cell lung cancer treatments (mNSCLC) and metastatic castration-resistant prostate cancer (mCRPC). Records were reviewed by humans and two AAMs. AAM-1 involved a pre-trained data-mining model specialized in biomedical literature, and AAM-2 was based on support vector machine algorithms. The AAMs assigned an accept/reject status, with reasons for exclusion. Automatic results were compared to those of humans. For mNSCLC, 5820 records were processed by humans and 440 (8%) records were accepted and the remaining items rejected. AAM-1 correctly accepted 6% of records and correctly excluded 79%. AAM-2 correctly accepted 6% of records and correctly excluded 82%. The review was completed by AAM-1 or AAM-2 in 52 hours, compared to 196 hours for humans. Work saved was estimated to be 76% and 79% by AAM-1 and AAM-2, respectively. For mCRPC, 2434 records were processed by humans and 26% of these were accepted and 74% rejected. AAM-1 correctly accepted 23% of records and rejected 62%. AAM-2 correctly accepted 20% of records and rejected 66%. The review was completed by AAM-1, AAM-2, and humans in 25, 25 and 85 hours, respectively. Work saved was estimated to be 61% and 68% by AAM-1 and AAM-2, respectively. AAMs can markedly reduce the time required for searching and triaging records during a systematic review. Methods similar to AAMs should be assessed in future research for how consistent their performances are in SLRs of economic, epidemiological and humanistic evidence.


title: Ready-to-eat foods: A potential vehicle for spread of coagulase-positive staphylococci and antibiotic-resistant Staphylococcus aureus in Buea municipality, South West Cameroon

abstract:
BackgroundThe consumption of ready-to-eat (RTE) foods contaminated with coagulase-positive staphylococci (CoPS) and especially Staphylococcus aureus puts consumers at potential risk of foodborne disease or colonization and subsequent infection. This cross-sectional study determined the levels of CoPS and the presence of S. aureus in RTE foods sold in Buea municipality.

MethodA total of 420 RTE food samples comprising 70 each of cake, bread, fruit salad, meat-hot-pot, suya and boiled rice were randomly purchased from February to August 2020. The CoPS counts were determined by culturing on Baird-Parker agar and S. aureus identified by amplification of the nuc gene using polymerase chain reaction. All S. aureus isolates were screened for the presence of classical staphylococcal enterotoxin genes and each isolate challenged with 11 antibiotics to determine their antibiotic susceptibility profiles. Oxacillin-resistant S. aureus were analyzed for the presence of mecA gene.

ResultOverall, 161 (38.3%) samples had detectable levels of coagulase-positive staphylococci ranging from 2.0-5.81 log10CFU/g. Based on CoPS levels, 37 (8.81%) of the 420 RTE food samples, only fruit salad and meat-hot-pot, had unsatisfactory microbiological quality. A total of 72 S. aureus isolates, comprising 52.78% from fruit salad, 16.67% from meat-hot-pot, 12.5% from boiled rice, 9.72% from suya, 5.56% from bread and 4.17% from cake were recovered. None of the S. aureus isolates possessed any of the classical enterotoxin genes. All the isolates were sensitive to vancomycin and ofloxacin while 68 (94.44%) and 66 (91.67%) were sensitive to oxacillin and ciprofloxacin, respectively. Resistance to penicillin (93.06%) was highest followed by amoxicillin (91.67%) and erythromycin (79.17%). Four isolates were identified as methicillin-resistant S. aureus all of which carried the mecA gene. A total of 24 antibiotypes were identified.

ConclusionOur findings showed that RTE foods sold in the Buea municipality are likely vehicles for transmission of CoPS and antibiotic-resistant S. aureus.


title: Competitive evolution of H1N1 and H3N2 influenza viruses in the United States

abstract: Seasonal influenza causes vast public health and economic impact globally. The prevention and control of the annual epidemics remain a challenge due to the antigenic evolution of the viruses. Here, we presented a novel modeling framework based on changes in amino acid sequences and relevant epidemiological data to retrospectively investigate the competitive evolution and transmission of H1N1 and H3N2 influenza viruses in the United States during October 2002 and April 2019. To do so, we estimated the time-varying disease transmission rate from the reported influenza cases and the time-varying antigenic change rate of the viruses from the changes in amino acid sequences. By incorporating the time-varying antigenic change rate into the transmission models, we found that the models could capture the evolutionary transmission dynamics of influenza viruses in the United States. Our modeling results also showed that the antigenic change of the virus plays an essential role in seasonal influenza dynamics.

category: evolutionary biology


title: Competitive evolution of H1N1 and H3N2 influenza viruses in the United States: A mathematical modeling study


title: New tools for automated cryo-EM single-particle analysis in RELION-4.0

abstract: 1We describe new tools for the processing of electron cryo-microscopy (cryo-EM) images in the fourth major release of the RELION software. In particular, we introduce VDAM, a Variable-metric gradient Descent algorithm with Adaptive Moments estimation, for image refinement; a convolutional neural network for unsupervised selection of 2D classes; and a flexible framework for the design and execution of multiple jobs in pre-defined workflows. In addition, we present a stand-alone utility called MDCatch that links the execution of jobs within this framework with metadata gathering during microscope data acquisition. The new tools are aimed at providing fast and robust procedures for unsupervised cryo-EM structure determination, with potential applications for on-the-fly processing and the development of flexible, high-throughput structure determination pipelines. We illustrate their potential on twelve publicly available cryo-EM data sets.


title: Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

abstract: Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, {pi}-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute {pi}-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, {pi}-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of {pi}-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.


title: Multiphoton microscopy for label-free multicolor imaging of peripheral nerve

abstract: Conventional histomorphometry of peripheral nerve entails lengthy chemical processing, ultrathin sectioning in resin, and imaging by light or electron microscopy. Multiphoton microscopy techniques exist enabling label-free and in vivo imaging of histological samples. Third-harmonic-generation microscopy has recently been demonstrated effective for imaging the myelin sheath of peripheral nerve axons in animal models. Herein, we characterize use of second and third harmonic generation microscopy for label-free imaging of murine and human peripheral nerve via a novel multicolor multiphoton microscope based on a single excitation wavelength at 1300 nm. Second harmonic generation signal from collagen centered about 650 nm delineates neural connective tissue, while third harmonic general signal centered about 433 nm delineates myelin and other lipids. In transgenic mice expressing yellow fluorescent protein linked to the thy1 promoter, three-photon-excitation with emission peak at 527 nm delineates axoplasm. We compare label-free multiphoton imaging of murine and human peripheral nerve against conventional chemical stains and discuss clinical implications of this approach in guiding intraoperative decision making in nerve transfer procedures.


title: An Evolutionarily Conserved Regulatory Pathway of Muscle Mitochondrial Network Organization

abstract: Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial network-type separately from contractile fiber-type remain unclear. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated independently. Proteomic analyses of indirect flight, jump, and leg muscles together with muscles misexpressing known fiber-type specification factor salm identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization separately from contractile type, mitochondrial content, and mitochondrial size through an evolutionarily conserved pathway involving cut, salm, and H15.


title: Cytoskeletal regulation of a transcription factor by DNA mimicry

abstract: A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. In contrast, the principal effectors of Hedgehog signaling, the Gli transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How Gli is tethered to microtubules remains unclear. We uncover DNA mimicry by the ciliary kinesin Kif7 as a mechanism for the recruitment of Gli to microtubules, revealing a new mode of tethering a DNA-binding protein to the cytoskeleton. Gli increases the Kif7-microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive Gli tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We re-tooled the unique DNA-mimicry-based Gli-Kif7 interaction for inhibiting the nuclear and cilium localization of Gli. This strategy can be potentially exploited for downregulating erroneously activated Gli in human cancers.


title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

abstract: Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.


title: Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts


title: In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture

abstract:
The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.

SummaryNuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the intact embryos and tissues of Drosophila melanogaster and its potential applications.


title: Progranulin deficiency results in reduced bis(monoacylglycero)phosphate (BMP) levels and gangliosidosis

abstract: Homozygous mutations of granulin precursor (GRN) lead to neuronal ceroid lipofuscinosis1, a severe neurodevelopmental disease, in humans and neuroinflammation in mice2. Haploinsufficiency of GRN almost invariably causes frontotemporal dementia (FTD)3,4. The GRN locus produces progranulin (PGRN), a lysosomal precursor protein that is cleaved to granulin peptides5,6. Despite intensive investigation, the function of granulins and the reason why their absence causes neurodegeneration remain unclear. Here, we investigated PGRN function in lipid degradation, a major function of lysosomes. We show that PGRN-knockout human cells, PGRN-deficient murine brain, and frontal lobes of human brains from patients with GRN mutation-related FTD have increased levels of gangliosides, highly abundant sialic acid-containing glycosphingolipids (GSL) that are degraded in lysosomes. Probing how PGRN deficiency causes these changes, we found normal levels and activities of enzymes that catabolize gangliosides. However, levels of bis(monoacylglycero)phosphate (BMP), a lysosomal lipid required for ganglioside catabolism7, were markedly reduced in PGRN-deficient cells and patient brain tissues. These data indicate that granulins are required to maintain BMP levels, which regulate ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. This aberrant accumulation of gangliosides may contribute to neuroinflammation and neurodegeneration susceptibility.


title: Loss of the GARP but not EARP protein complex drives Golgi sterol overload during dendrite remodeling

abstract: Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.


title: A role for cell polarity in lifespan and mitochondrial quality control in the budding yeast Saccharomyces cerevisiae

abstract: Babies are born young, largely independent of the age of their mothers. Mother-daughter age asymmetry in yeast is achieved, in part, by inheritance of higher-functioning mitochondria by daughter cells and retention of some high-functioning mitochondria in mother cells. The mitochondrial F-box protein, Mfb1p, tethers mitochondria at both poles in a cell cycle-regulated manner: it localizes to and anchors mitochondria to the mother cell tip throughout the cell cycle, and to the bud tip prior to cytokinesis. Here, we report that cell polarity and polarized localization of Mfb1p decline with age in S. cerevisiae. Moreover, deletion of BUD1/RSR1, a Ras protein required for cytoskeletal polarization during asymmetric yeast cell division, results in depolarized Mfb1p localization, defects in mitochondrial distribution and quality control, and reduced replicative lifespan. Our results demonstrate a role for the polarity machinery in lifespan through modulating Mfb1 function in asymmetric inheritance of mitochondria during yeast cell division.


title: Efficient Fluorescence-Based Localization Technique for Tracking Endophytes Route in Host-Plants Colonization

abstract:
Bacterial isolates that enhance plant growth and suppress plant pathogens growth are essential tools for reducing pesticide applications in plant production systems. The objectives of this study were to develop a reliable fluorescence-based technique for labeling bacterial isolates selected as biological control agents (BCAs) to allow their direct tracking in the host-plant interactions, understand the BCA localization within their host plants, and the route of plant colonization. Objectives were achieved by developing competent BCAs transformed with two plasmids, pBSU101 and pANIC-10A, containing reporter genes eGFP and pporRFP, respectively. Our results revealed that the plasmid-mediated transformation efficiencies of antibiotic-resistant competent BCAs identified as PSL, IMC8, and PS were up 84%. Fluorescent BCA-tagged reporter genes were associated with roots and hypocotyls but not with leaves or stems and were confirmed by fluoresence microscopy and PCR analyses in colonized Arabidopsis and sorghum. This fluorescence-based techniques high resolution and reproducibility make it a platform-independent system that allows tracking of BCAs spatially within plant tissues, enabling assessment of the movement and niches of BCAs within colonized plants. Steps for producing and transforming competent fluorescent BCAs, as well as the inoculation of plants with transformed BCAs, localization, and confirmation of fluorescent BCAs through fluorescence imaging and PCR, are provided in this manuscript. This study features host-plant interactions and subsequently biological and physiological mechanisms implicated in these interactions. The maximum time to complete all the steps of this protocol is approximately three months.

SENTENCE SUMMARYWe describe a novel fluorescence localization technique as a powerful tool to directly visualize and determine the route in-situ of BCAs in host-plants interaction. The study features the host-plant interactions, biological and physiological responses implicated.


title: Anisotropy of plasmalemmal sterols and cell mating require StARkin domain proteins Ysp2 and Lam4.

abstract:
In the budding yeast S. cerevisiae Cdc42 is required for polarized growth and the formation of mating projections (shmoos). Negatively charged lipids including phosphatidylserine and phosphatidylinositol 4,5-bisphosphate support a positive feedback loop that recruits Cdc42 effectors and MAP kinase scaffolds, many of which contain polybasic patches that directly interact with the membrane. Here, using genetically encoded sterol sensor ALOD4 we find that ergosterol is accumulated in the cytosolic leaflet of buds and shmoos. The accumulation of ergosterol in the plasma membrane requires both Osh and Lam proteins however cells lacking Ysp2/Lam2 and Lam4 displayed a reversal in the polarity of ergosterol. The redistribution of ergosterol impairs the polarization of phosphatidylserine and phosphatidylinositol 4,5-bisphophate which further impacts shmoo formation, MAPK signaling and mating efficiency. Our observations demonstrate that the ability of Lam proteins to deliver ergosterol from the plasma membrane to the ER helps maintain a gradient of ergosterol which in turn supports robust cell polarity.

SummaryThe sterol sensor ALOD4 is enriched at sites of polarized growth. Elimination of the Osh proteins solubilized the ALOD4 whereas elimination of Ysp2 and Lam4 reversed ALOD4 polarization. Cells lacking Ysp2 and Lam4 have defects in mating and MAP kinase signaling.


title: Bayesian machine learning analysis of single-molecule fluorescence colocalization images

abstract: Multi-wavelength single-molecule fluorescence colocalization (CoSMoS) methods allow elucidation of complex biochemical reaction mechanisms. However, analysis of CoSMoS data is intrinsically challenging because of low image signal-to-noise ratios, non-specific surface binding of the fluorescent molecules, and analysis methods that require subjective inputs to achieve accurate results. Here, we use Bayesian probabilistic programming to implement Tapqir, an unsupervised machine learning method that incorporates a holistic, physics-based causal model of CoSMoS data. This method accounts for uncertainties in image analysis due to photon and camera noise, optical non-uniformities, non-specific binding, and spot detection. Rather than merely producing a binary "spot/no spot" classification of unspecified reliability, Tapqir objectively assigns spot classification probabilities that allow accurate downstream analysis of molecular dynamics, thermodynamics, and kinetics. We both quantitatively validate Tapqir performance against simulated CoSMoS image data with known properties and also demonstrate that it implements fully objective, automated analysis of experiment-derived data sets with a wide range of signal, noise, and non-specific binding characteristics.


title: Identification of CD73 as the antigen of an antigen-unknown monoclonal antibody established by exosome immunization, and its antibody-drug conjugate exerts an antitumor effect on glioblastoma cell lines

abstract: Development of antibodies against the native structure of membrane proteins with multiple transmembrane domains is challenging because it is difficult to prepare antigens with native structures. Previously, we successfully developed a monoclonal antibody against multi-pass membrane protein TMEM180 by exosome immunization in rats. This approach yielded antibod-ies that recognized cancer-specific antigens on the exosome. In this study, we performed im-munoprecipitation using magnetic beads to identify the antigen of one of the rat antibody clones, 0614, as CD73. We then converted antibody 0614 to human chimeric antibody 0614-5 and found that the humanized antibody did not inhibit CD73 enzymatic activity. Glioblastoma (GB) was the cancer type with the highest expression of CD73 in the tumor relative to healthy tissue. An antibody-drug conjugate (ADC) of 0614-5 exerted an antitumor effect on GB cell lines according to expression of CD73. The 0614-5-ADC could be used to treat cancers with high CD73 expression. In addition, our strategy could be used to determine the antigen of any antibody produced by exosome immunization.


title: Simulation of receptor triggering by kinetic segregation shows role of oligomers and close-contacts

abstract:
The activation of T cells, key players of the immune system, involves local evacuation of phosphatase CD45 from a region of the T cells surface, segregating it from the T cell receptor. What drives this evacuation? In the presence of antigen, what ensures evacuation happens in the sub-second timescales necessary to initiate signaling? In the absence of antigen, what mechanisms ensure evacuation does not happen spontaneously, which could cause signaling errors? Phenomena known to influence spatial organization of CD45 or similar surface molecules include diffusive motion in the lipid bilayer, oligomerization reactions, and mechanical compression against a nearby surface, such as that of the cell presenting antigen. Computer simulations can investigate hypothesized spatiotemporal mechanisms of T cell signaling. The challenge to computational studies of evacuation is that the base process, spontaneous evacuation by simple diffusion, is in the extreme rare event limit, meaning direct stochastic simulation is unfeasible. Here we combine particle-based spatial stochastic simulation with the Weighted Ensemble method for rare events to compute the mean first-passage time for cell surface availability by surface reorganization of CD45. We confirm mathematical estimates that, at physiological concentrations, spontaneous evacuation is extremely rare, roughly 300 years. We find that dimerization decreases the time required for evacuation. A weak bi-molecular interaction (dissociation constant estimate 460 microMolar) is sufficient for an order of magnitude reduction of spontaneous evacuation times, and oligomerization to hexamers reduces times to below 1 second. This introduces a mechanism whereby CD45 oligomerization could be accessible to an engineered therapeutic. For large regions of close-contact, such as those induced by large microvilli, molecular size and compressibility imply a nonzero re-entry probability 60%, decreasing evacuation times. Simulations show that these reduced evacuation times are still unrealistically long, suggesting that a yet-to-be-described mechanism, besides compressional exclusion at a close contact, drives evacuation.

Statement of SignificanceIn the immune system, T cells sensing pathogens depends on a process called T cell receptor triggering. In this process, proteins on the cell surface undergo reorganization, including local depletion of large membrane proteins from the area surrounding the T cell receptor. Computer simulations of protein dynamics provide a means to investigate phenomena in greater detail than that afforded by experiments. However, even simulations present challenges, because tracking the motion and interactions of individual molecules is computationally expensive. Combining a rare event algorithm with spatial simulations, we show that biochemical and mechanical properties drastically affect depletion timescales, and thus receptor triggering. Quantitative understanding of these timescales will constrain hypothesized mechanistic models and could suggest new strategies for T cell engineering.


title: Recombinant Fsh and Lh therapy for spawning induction of previtellogenic and early spermatogenic arrested teleost, the flathead grey mullet (Mugil cephalus)

abstract: With the expansion and diversification of global aquaculture, efforts continue to develop new bio-technologies for assisted reproduction in species that present reproductive dysfunctions. Flathead grey mullet (Mugil cephalus) held in intensive conditions in the Mediterranean region, display a severe reproductive dysfunction, where males do not produce fluent milt and females are arrested at previtellogenesis or early stages of vitellogenesis. In the present study, weekly injections of species-specific single-chain recombinant gonadotropins (rGths); follicle stimulating hormone (rFsh) (6 to 12 g kg-1 doses) and luteinizing hormone (rLh) (2.5 to 24 g kg-1 doses) were administered to induce vitellogenesis, from previtellogenesis / early vitellogenesis to the completion of vitellogenic growth in females and enhance spermatogenesis to produce adequate volumes of sperm from non-fluent males. During the experiment, all treated females (n = 21) developed oocytes in late vitellogenesis with 603 {+/-} 8 m diameter and all treated males produced fluent sperm. To induce oocyte maturation, ovulation and spawning, females were treated with either (i) a priming dose of 30 g kg-1 of rLh and a resolving dose of 40 mg kg-1 of progesterone (P4), (ii) priming and resolving doses of 30 g kg-1 of rLh, or (iii) priming and resolving doses of 40 mg kg-1 of P4 given 24:05 {+/-} 0:40 h apart. Females were placed in spawning tanks with rGth treated males that had fluent sperm. Spontaneous spawns of fertilised eggs were obtained after inducing with rLh + P4 or rLh + rLh (priming and resolving injections) with a spawning success of the 85% (8 of 9 females) and 100% (n = 6), respectively. The eggs collected from the tanks presented 64 {+/-} 22% fertilization with embryo development and 57 {+/-} 24 % hatching. The treatment P4 + P4 had a lower ovulation success (50 % - 3 of 6 females) and spawning success (17 %) with no fertilised eggs. Success was independent of the initial gonadal stage of females. In comparison, control females did not show any advance in gonadal development from initial stages and control males did not produce fluent sperm. The present results confirm the possibility of controlling oogenesis from previtellogenesis to the completion of maturation and fertilised tank spawning using exclusively rFsh and rLh in a teleost species.

category: physiology


title: Fast and ultra-sensitive glycoform analysis by supercritical fluid chromatography-tandem mass spectrometry

abstract: Therapeutic monoclonal antibodies (mAbs) are currently the largest and fastest growing category of biopharmaceuticals. Glycosylation of mAbs has a significant impact on their effector functions, as well as on their safety and pharmacokinetics. Heterogeneity of glycoforms is affected by various factors such as the producing cells and cell culture process. Therefore, accurate glycoform characterization is essential for drug design, process optimization, manufacturing, and quality control of therapeutic mAbs. In this study, we developed a fast, quantitative, and highly sensitive analytical platform for mAb glycan profiling by supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). An 8-minute analysis of bevacizumab, nivolumab, ramucirumab, rituximab, and trastuzumab by SFC-MS detected a total of 102 glycoforms, with a detection limit of 5 attomole. The dynamic range of glycan abundance was over 6 orders of magnitude for bevacizumab analysis by SFC-MS, compared to 3 orders of magnitude for conventional fluorescence HPLC analysis. This method revealed the glycan profile characteristics and lot-to-lot heterogeneity of various therapeutic mAbs. We were also able to detect a series of structural variations in pharmacologically important glycan structures. SFC-MS-based glycoform profiling method will provide an ideal platform for in-depth analysis of precise glycan structure and abundance.


title: Simulations of flow over a bio-inspired undulated cylinder with dynamically morphing topography

abstract: Inspired by the geometric properties of seal whiskers, the addition of spanwise undulations on a cylinder have been shown to lower the mean and oscillatory forces and modify the frequency of flow-induced vibration when compared to smooth cylinders. Previously, computational fluid dynamics (CFD) has been used to characterize the hydrodynamic response with respect to specific geometric features. However simulations are time intensive due to complex three-dimensional meshing and computation time, limiting the number of geometric perturbations explored. A method is proposed in which the specific geometric features of this complex topography can be modified during a simulation thereby decreasing the time per geometric modification and removing the need for manual meshing of the complex structure. The surface of the seal whisker inspired geometry is parameterized into seven defining parameters, each of which is directly controlled in order to morph the surface into any realization within the defined parameter space. Once validated, the algorithm is used to explore the undulation amplitudes in the chord and thickness directions, by varying each independently from 0 to 0.3 thicknesses in increments of 0.05 at a Reynolds number of 500. The force and frequency response are examined for this matrix of geometric parameters, yielding detailed force trends not previously investigated. The impact of the bio-inspired morphing algorithm will allow for further optimization and development of force-mitigating underwater devices and other engineering applications in need of vibration suppression, frequency tuning, or force reduction.


title: Re-charging your fats: Charmm36 parameters for neutral lipids triacylglycerol and diacylglycerol

abstract: Neutral lipids (NLs) are an abundant class of cellular lipids. They are characterized by the total lack of charged chemical groups in their structure, and, as a consequence, they play a major role in intracellular lipid storage. NLs that carry a glycerol backbone, such as triacylglycerols (TGs) and diacylglycerols (DGs), are also involved in the biosynthetic pathway of cellular phospholipids, and they have recently been the subject of numerous structural investigations by means of atomistic molecular dynamics (MD) simulations. However, conflicting results on the physicochemical behavior of NLs were observed depending on the nature of the atomistic force field used. Here, we show that current phospholipid-derived CHARMM36 parameters for DGs and TGs cannot reproduce adequately interfacial properties of these NLs, due to excessive hydrophilicity at the glycerol-ester region. By following a CHARMM36-consistent parameterization strategy, we develop new parameters for both TGs and DGs that are compatible with both cutoffbased and Particle Mesh Ewald (PME) schemes for the treatment of Lennard Jones interactions. We show that our new parameters can reproduce interfacial properties of NLs and their behavior in more complex lipid assemblies. We discuss the implications of our findings in the context of intracellular lipid storage and NLs cellular activity.


title: Transferability of Geometric Patterns from Protein Self-Interactions to Protein-Ligand Interactions

abstract: There is significant interest in developing machine learning methods to model protein-ligand interactions but a scarcity of experimentally resolved protein-ligand structures to learn from. Protein self-contacts are a much larger source of structural data that could be leveraged, but currently it is not well understood how this data source differs from the target domain. Here, we characterize the 3D geometric patterns of protein self-contacts as probability distributions. We then present a flexible statistical framework to assess the transferability of these patterns to protein-ligand contacts. We observe that the level of transferability from protein self-contacts to protein-ligand contacts depends on contact type, with many contact types exhibiting high transferability. We then demonstrate the potential of leveraging information from these geometric patterns to aid in ligand pose-selection problems in protein-ligand docking. We publicly release our extracted data on geometric interaction patterns to enable further exploration of this problem.