title: Ion channel expression and electrophysiology of singular human (primary and induced pluripotent stem cell derived) cardiomyocytes

abstract: SUMMARYHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and primary human cardiomyocytes are used for in vitro cardiac safety testing. hiPSC-CMs have been associated with a vast heterogeneity regarding single-cell morphology, beating behavior and action potential duration, prompting a systematic analysis of single-cell characteristics. Previously published hiPSC-CM studies revealed action potentials with nodal-, atrial- or ventricular-like morphology, although ion channel expression of singular hiPSC-CMs is not fully understood. Other studies used single-cell RNA-sequencing, however, these studies did not extensively focus on expression patterns of cardiac ion channels or failed to detect ion channel transcripts. Thus, the current study used a single-cell patch-clamp-RT-qPCR approach to get insights into single-cell electrophysiology (capacitance, action potential duration at 90% of repolarization, upstroke velocity, spontaneous beat rate, and sodium-driven fast inward current) and ion channel expression (HCN4, CACNA1G, CACNA1D, KCNA5, KCNJ4, SCN5A, KCNJ2, CACNA1D, and KCNH2), the combination of both within individual cells, and their correlations in single cardiomyocytes. We used commercially available hiPSC-CMs (iCell cardiomyocytes, atrial and ventricular Pluricytes) and primary human adult atrial and ventricular cardiomyocytes. Recordings of electrophysiological parameters revealed differences between the cell groups and variation within the hiPSC-CMs groups as well as within primary ventricular cardiomyocytes. Expression analysis on mRNA level showed no-clear-cut discrimination between primary cardiac subtypes and revealed both similarities and differences between all cell groups. Higher expression of atrial-associated ion channels in primary atrial cardiomyocytes and atrial Pluricytes compared to their ventricular counterpart indicates a successful chamber-specific hiPSC differentiation. Interpretation of correlations between the single-cell parameters was challenging, as the total data set is complex, particularly for parameters depending on multiple processes, like the spontaneous beat rate. Yet, for example, expression of SCN5A correlated well with the fast inward current amplitude for all three hiPSC-CM groups. To further enhance our understanding of the physiology and composition of the investigated hiPSC-CMs, we compared beating and non-beating cells and assessed distributions of single-cell data. Investigating the single-cell phenotypes of hiPSC-CMs revealed a combination of attributes which may be interpreted as a mixture of traits of different adult cardiac cell types: (i) nodal-related pacemaking attributes are spontaneous generation of action potentials and high HCN4 expression; and (ii) non-nodal attributes: cells have a prominent INa-driven fast inward current, a fast upstroke velocity and a high expression of SCN5A. In conclusion, the combination of nodal- and non-nodal attributes in single hiPSC-CMs may hamper the interpretation of drug effects on complex electrophysiological parameters like beat rate and action potential duration. However, the proven expression of specific ion channels enables the evaluation of drug effects on ionic currents in a more realistic environment than in recombinant systems.

category: pharmacology and toxicology


title: Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

abstract: In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

category: immunology


title: SARS-CoV-2 variant with higher affinity to ACE2 shows reduced sera neutralization susceptibility

abstract:
BackgroundSeveral new variants of SARS-CoV-2 have emerged since fall 2020 which have multiple mutations in the receptor binding domain (RBD) of the spike protein.

ObjectiveWe aimed to assess how mutations in the SARS-CoV-2 RBD affect receptor affinity to angiotensin-converting enzyme 2 (ACE2) and neutralization by anti-RBD serum antibodies.

MethodsWe produced a SARS-CoV-2 RBD mutant (RBDmut) with key mutations (E484K, K417N, N501Y) from the newly emerged Brazilian variant. Using Biolayer Interferometry, we analyzed the binding of this mutant to ACE2, and the susceptibility to neutralization by sera from vaccinated mice and COVID-19 convalescent patients.

ResultsKinetic profiles showed increased RBDmut - ACE2 affinity compared to RBDwt, and binding of vaccine-elicited or convalescent sera was significantly reduced. Likewise, both sera types showed significantly reduced ability to block RBDmut - ACE2 binding indicating that antibodies induced by RBDwt have reduced capability to neutralize mutant virus.

ConclusionOur physiochemical data show enhanced infectivity and reduced neutralization by polyclonal antibodies of the Brazilian variant of SARS-CoV-2.

Capsule summarySARS-CoV-2 variant with Brazilian RBD mutations shows increased ACE2 affinity and reduced susceptibility to blockage by vaccine-elicited and convalescent sera.


title: The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory

abstract:
Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against coronavirus disease in 2019 (COVID-19). Clinical trials and ongoing vaccinations present with very high protection levels and varying degrees of side effects. However, the nature of the reported side effects remains poorly defined. Here we present evidence that LNPs used in many preclinical studies are highly inflammatory in mice. Intradermal injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate.

In summary, here we show that the LNPs used for many preclinical studies are highly inflammatory. Thus, their potent adjuvant activity and reported superiority comparing to other adjuvants in supporting the induction of adaptive immune responses likely stem from their inflammatory nature. Furthermore, the preclinical LNPs are similar to the ones used for human vaccines, which could also explain the observed side effects in humans using this platform.


title: SARS-CoV-2 viability in time on experimental surfaces

abstract: We evaluated the SARS-CoV-2 viability preservation on different model surfaces over time. It was found that the SARS-CoV-2 RNA was detected on all studied surfaces for 360 minutes, while the viability of the virus was completely lost after 120 minutes. Type of experimental surface significantly affects viability preservation.

category: microbiology


title: Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation

abstract: There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3 antisense sequence for target recognition and a 5-phosphorylated 2-5 poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or {Delta}H69/{Delta}V70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.


title: Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal

abstract: Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including CSE, FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

category: molecular biology


title: Sod1 Integrates Oxygen Availability to Redox Regulate NADPH Production and the Thiol Redoxome

abstract:
Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2-) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major new aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is exclusively dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2- to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this new mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2- toxicity, which only requires < 1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Significance StatementCu/Zn superoxide dismutase (Sod1) is a key antioxidant enzyme and its importance is underscored by the fact that its ablation in cell and animal models results in oxidative stress, metabolic defects, and reductions in cell proliferation, viability, and lifespan. Curiously, Sod1 detoxifies superoxide radicals (O2-) in a manner that produces an oxidant as a byproduct, hydrogen peroxide (H2O2). While much is known about the necessity of scavenging O2-, it is less clear what the physiological roles of Sod1-derived H2O2 are. Herein, we discovered that Sod1-derived H2O2 plays a very important role in antioxidant defense by stimulating the production of NADPH, a vital cellular reductant required for ROS scavenging enzymes, as well as redox regulating a large network of enzymes.

category: biochemistry


title: Structural insights into plasticity and discovery of remdesivir metabolite GS-441524 binding in SARS-CoV-2 macrodomain

abstract: The nsP3 macrodomain is a conserved protein interaction module that plays essential regulatory roles in host immune response by recognizing and removing posttranslational ADP-ribosylation sites during SARS-CoV-2 infection. Thus, targeting this protein domain may offer a therapeutic strategy to combat the current and future virus pandemics. To assist inhibitor development efforts, we report here a comprehensive set of macrodomain crystal structures complexed with diverse naturally-occurring nucleotides, small molecules as well as nucleotide analogues including GS-441524 and its phosphorylated analogue, active metabolites of remdesivir. The presented data strengthen our understanding of the SARS-CoV-2 macrodomain structural plasticity and it provides chemical starting points for future inhibitor development.


title: A novel conformational state for SARS-CoV-2 main protease

abstract: The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of coronavirus, making it a promising target for drugs against Covid-19 pandemic. Here we present a crystal structure of Mpro disclosing new structural features of key regions of the enzyme. We show that the oxyanion loop, involved in substrate recognition and enzymatic activity, can adopt a new conformation, which is stable and significantly different from the known ones. In this new state the S1 subsite of the substrate binding region is completely reshaped and a new cavity near the S2 subsite is created. This new structural information expands the knowledge of the conformational space available to Mpro, paving the way for the design of novel classes of inhibitors specifically designed to target this unprecedented binding site conformation, thus enlarging the chemical space for urgent antiviral drugs against Covid-19 pandemic.


title: Diffusion barriers and adaptive carbon uptake strategies enhance the modeled performance of the algal CO2-concentrating mechanism

abstract:
Many photosynthetic organisms enhance the performance of their CO2-fixing enzyme Rubisco by operating a CO2-concentrating mechanism (CCM). Most CCMs in eukaryotic algae supply concentrated CO2 to Rubisco in an organelle called the pyrenoid. Ongoing efforts seek to engineer an algal CCM into crops that lack a CCM to increase yields. To advance our basic understanding of the algal CCM, we develop a chloroplast-scale reaction-diffusion model to analyze the efficacy and the energy efficiency of the CCM in the green alga Chlamydomonas reinhardtii. We show that achieving an effective and energetically efficient CCM requires a physical barrier such as thylakoid stacks or a starch sheath to reduce CO2 leakage out of the pyrenoid matrix. Our model provides insights into the relative performance of two distinct inorganic carbon uptake strategies: at air-level CO2, a CCM can operate effectively by taking up passively diffusing external CO2 and catalyzing its conversion to HCO3-, which is then trapped in the chloroplast; however, at lower external CO2 levels, effective CO2 concentration requires active import of HCO3-. We also find that proper localization of carbonic anhydrases can reduce futile carbon cycling between CO2 and HCO3-, thus enhancing CCM performance. We propose a four-step engineering path that increases predicted CO2 saturation of Rubisco up to seven-fold at a theoretical cost of only 1.5 ATP per CO2 fixed. Our system-level analysis establishes biophysical principles underlying the CCM that are broadly applicable to other algae and provides a framework to guide efforts to engineer an algal CCM into land plants.

Significance StatementEukaryotic algae mediate approximately one-third of CO2 fixation in the global carbon cycle. Many algae enhance their CO2-fixing ability by operating a CO2-concentrating mechanism (CCM). Our model of the algal CCM lays a solid biophysical groundwork for understanding its operation. The models consistency with experimental observations supports existing hypotheses about the operating principles of the algal CCM and the functions of its component proteins. We provide a quantitative estimate of the CCMs energy efficiency and compare the performance of two distinct CO2 assimilation strategies under varied conditions. The model offers a quantitative framework to guide the engineering of an algal CCM into land plants and supports the feasibility of this endeavor.

category: biophysics


title: The NADPH oxidase NOX2 is a marker of adverse prognosis involved in chemoresistance of Acute Myeloid Leukemias

abstract: Resistance to chemotherapeutic drugs is a major cause of treatment failure in Acute Myeloid Leukemias (AML). To better characterize the mechanisms of chemoresistance, we first identified genes whose expression is dysregulated in AML cells resistant to daunorubicin (DNR) or cytarabine (Ara-C), the main drugs used for the induction therapy. The genes found activated are mostly linked to immune signaling and inflammation. Among them, we identified a strong up-regulation of the NOX2 NAPDH oxidase subunit genes (CYBB, CYBA, NCF1, NCF2, NCF4 and RAC2). The ensuing increase in NADPH oxidase activity, which is particularly strong in DNR-resistant cells, participates in the acquisition and/or maintenance of resistance to DNR. In addition, analyzing gp91phox (CYBB-encoded Nox2 catalytic sub-unit) expression at the surface of leukemic blasts from 74 patients at diagnosis showed that NOX2 is generally more expressed and active in leukemic cells from the FAB M4/M5 subtypes compared to FAB M0-M2 ones. Using a gene expression-based score we demonstrate that high NOX2 subunit genes expression is a marker of adverse prognosis, independent of the cytogenetic-based risk classification, in AML patients.

category: cancer biology


title: Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

abstract:
ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells.

MethodsFresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 h. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations and single-cell RNA-seq, and ELISA and LEGENDplex analysis of PsA SF were also performed.

ResultsWe observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be amongst those most highly upregulated by PsA monocytes/macrophages; and both proteins were elevated in PsA SF.

ConclusionsUsing multiomic analyses we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.


title: UBXN3B Restricts Viral Pathogenesis by Maintaining Hematopoietic Homeostasis

abstract: Hematopoiesis is finely regulated to enable timely production of the right numbers and types of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential role of UBXN3B in maintenance of hematopoietic homeostasis and restriction of immunopathogenesis during respiratory viral infection. Ubxn3b deficient (Ubxn3b-/-) mice are highly vulnerable to SARS-CoV-2 and influenza A infection, characterized by more severe lung immunopathology, lower virus-specific IgG, significantly fewer B cells, but more myeloid cells than Ubxn3b+/+ littermates. This aberrant immune compartmentalization is recapitulated in uninfected Ubxn3b-/- mice. Mechanistically, UBXN3B controls precursor B-I (pre-BI) transition to pre-BII and subsequent proliferation in a cell-intrinsic manner, by maintaining BLNK protein stability and pre-BCR signaling. These results reveal an essential role of UBXN3B for the early stage of B cell development.


title: An Essential Role of UBXN3B for B Lymphopoiesis


title: An Essential Role of UBXN3B in B Lymphopoiesis


title: T-cell Repertoire Characteristics of Asymptomatic and Re-detectable Positive COVID-19 Patients

abstract:
BackgroundThe prevention of COVID-19 pandemic is highly complicated by the prevalence of asymptomatic and recurrent infection. Many previous immunological studies have focused on symptomatic and convalescent patients, while the immune responses in asymptomatic patients and re-detectable positive cases remain unclear.

MethodsHere we comprehensively analyzed the peripheral T-cell receptor (TCR) repertoire of 54 COVID-19 patients in different phases, including asymptomatic, symptomatic, convalescent and re-detectable positive cases.

ResultsWe found progressed immune responses from asymptomatic to symptomatic phase. Furthermore, the TCR profiles of re-detectable positive cases were highly similar to those of asymptomatic patients, which could predict the risk of recurrent infection.

ConclusionTherefore, TCR repertoire surveillance has the potential to strengthen the clinical management and the immunotherapy development for COVID-19.

FundingThe Science and Technology Innovation Project of Foshan Municipality (2020001000431) and the National Key Research and Development Project (2020YFA0708001).


title: Accounting for B cell behaviour and sampling bias yields a superior predictor of anti-PD-L1 response in bladder cancer

abstract:
Tumor-infiltrating B cells and intratumorally-produced immunoglobulins (IG) play important roles in the tumor microenvironment and response to immunotherapy1-5. IgG antibodies produced by intratumoral B cells may drive antibody-dependent cellular cytotoxicity (ADCC) and enhance antigen presentation by dendritic cells6-8. Furthermore, B cells are efficient antigen-specific antigen presenters that can essentially modulate the behaviour of helper T cells9-11.

Here we investigated the role of intratumoral IG isotype and clonality in bladder cancer. Our results show that the IgG1/IgA ratio offers a strong and independent prognostic indicator for the Basal squamous molecular subtype and for the whole ImVigor210 cohort in anti-PD-L1 immunotherapy. Our findings also indicate that effector B cell functions, rather than clonally-produced antibodies, are involved in the antitumor response. High IgG1/IgA ratio was associated with relative abundance of cytotoxic genes and prominence of the IL-21/IL-21R axis suggesting importance of T cell/B cell interaction.

We integrated the B, NK, and T cell components, employing immFocus-like normalization to account for the stochastic nature of tumor tissue sampling. Using a random forest model with nested cross-validation, we developed a tumor RNA-Seq-based predictor of anti-PD-L1 therapy response in muscle-invasive urothelial carcinoma. The resulting PRIMUS (PRedIctive MolecUlar Signature) predictor achieves superior sensitivity compared to PD-L1 expression scores or existing gene signatures, allowing for reliable identification of responders even within the desert patient subcohort analyzed as a hold out set.


title: The memory B cell response to influenza vaccination is impaired in older persons

abstract:
Influenza imparts an age-related increase in mortality and morbidity. The most effective countermeasure is vaccination; however, vaccines offer modest protection in older adults. To investigate how ageing impacts the memory B cell response we tracked haemagglutinin specific B cells by indexed flow sorting and single cell RNA sequencing in twenty healthy adults administered the trivalent influenza vaccine. We found age-related skewing in the memory B cell compartment six weeks after vaccination, with younger adults developing haemagglutinin specific memory B cells with an FCRL5+ "atypical" phenotype, showing evidence of somatic hypermutation and positive selection, which happened to a lesser extent in older persons. We confirmed the germinal center ancestry of these FCRL5+ "atypical" memory B cells using scRNASeq from fine needle aspirates of influenza responding human lymph nodes and paired blood samples. Together, this study shows that the aged human germinal center reaction and memory B cell response following vaccination is defective.

SummaryImmune responses to vaccination wane with age. Using single cell RNA sequencing of influenza vaccine specific B cells, this study delineates changes in B cell memory generation, antibody mutation and their subsequent selection in older persons.


title: Combination of in vivo phage therapy data with in silico model highlights key parameters for treatment efficacy

abstract: The clinical (re)development of phage therapy to treat antibiotic resistant infections requires grasping specific biological properties of bacteriophages (phages) as antibacterial. However, identification of optimal dosing regimens is hampered by the poor understanding of phage-bacteria interactions in vivo. Here we developed a general strategy coupling in vitro and in vivo experiments with a mathematical model to characterize the interplay between phage and bacterial dynamics during pneumonia induced by a pathogenic strain of Escherichia coli. The model estimates some key parameters for phage therapeutic efficacy, in particular the impact of dose and route of administration on phage dynamics and the synergism of phage and the innate immune response on the bacterial clearance rate. Simulations predict a low impact of the intrinsic phage characteristics in agreement with the current semi-empirical choices of phages for compassionate treatments. Model-based approaches will foster the deployment of future phage therapy clinical trials.


title: Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

abstract:
In bacteria, the nucleotide-based second messenger bis-(3-5)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility.

Importancec-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts upstream of the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts downstream of the Frz system to regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple parallel pathways to regulate motility and development in M. xanthus.


title: Disinfection of SARS-CoV-2 contaminated surfaces of personal items with UVC-LED disinfection boxes

abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted from person-to-person by close contact, small aerosol respiratory droplets and potentially via contact with contaminated surfaces. Here, we investigated the effectiveness of commercial UVC-LED disinfection boxes in inactivating SARS-CoV-2 contaminated surfaces of personal items. We contaminated glass, metal and plastic samples representing the surfaces of personal items such as smartphones, coins or credit cards with SARS-CoV-2 formulated in an organic matrix mimicking human respiratory secretions. For disinfection, the samples were placed at different distances from UVC emitting LEDs inside commercial UVC-LED disinfection boxes and irradiated for different time periods (up to 10 minutes). High viral loads of SARS-CoV-2 were effectively inactivated on all surfaces after 3 minutes of irradiation. Even 10 seconds of UVC-exposure strongly reduced viral loads. Thus, UVC-LED boxes proved to be an effective method for disinfecting SARS-CoV-2 contaminated surfaces that are typically found on personal items.


title: Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the feces of a wild blackbuck antelope

abstract: The anaerobic gut fungi (AGF, phylum Neocallimastigomycota) reside in the alimentary tracts of herbivores. Multiple novel, yet-uncultured AGF taxa have recently been identified in culture-independent diversity surveys. Here, we report on the isolation and characterization of the first representative of the RH5 lineage from fecal samples of a wild blackbuck (Indian Antelope) from Sutton County, Texas, USA. The isolates displayed medium sized (2-4 mm) compact circular colonies on agar roll tubes and thin loose biofilm-like growth in liquid medium. Microscopic examination revealed monoflagellated zoospores and polycentric thalli with highly branched nucleated filamentous rhizomycelium, a growth pattern encountered in a minority of described AGF genera so far. The obtained isolates are characterized by formation of spherical vesicles at the hyphal tips from which multiple sporangia formed either directly on the spherical vesicles or at the end of sporangiophores. Phylogenetic analysis using the D1/D2 regions of the large ribosomal subunit (D/D2 LSU) and the ribosomal internal transcribed spacer 1 (ITS1) revealed sequence similarities of 93.5%, and 81.3%, respectively, to the closest cultured relatives (Orpinomyces joyonii strain D3A (D1/D2 LSU), and Joblinomyces apicalis strain GFH681 (ITS1). Substrate utilization experiments using the type strain (BB-3) demonstrated growth capabilities on a wide range of mono-, oligo-, and polysaccharides, including glucose, xylose, mannose, fructose, cellobiose, sucrose, maltose, trehalose, lactose, cellulose, xylan, starch, and raffinose. We propose accommodating these novel isolates in a new genus and species, for which the name Paucimyces polynucleatus is proposed. The type species is strain BB-3.


title: The Borrelia burgdorferi adenylyl cyclase, CyaB, is important for virulence factor production and mammalian infection

abstract: Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3 end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full length SR0623 to evaluate the influence on transcriptional and post-transcriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.


title: Stimulation of adaptive gene amplification by origin firing under replication fork constraint

abstract: Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32, factors known to alter the frequency of template switching during break induced replication (BIR). CUP1 amplification is also critically dependent on late firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after firing, leaving an epigenetic scar of H3K56 acetylation that promotes template switching during later break induced replication events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.


title: Locus specific epigenetic modalities of random allelic expression imbalance

abstract: Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression was not observed in ESCs, but when we assessed expression status in more than 200 clones of neuronal progenitor cells, we observed high frequencies of monoallelism. We uncovered unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up novel therapeutic perspectives.


title: The genetics of gene expression in a C. elegans multi parental recombinant inbred line population.

abstract: Studying genetic variation of gene expression provides a powerful way to unravel the molecular components underlying complex traits. Expression QTL studies have been performed in several different model species, yet most of these linkage studies have been based on genetic segregation of two parental alleles. Recently we developed a multi-parental segregating population of 200 recombinant inbred lines (mpRILs) derived from four wild isolates (JU1511, JU1926, JU1931 and JU1941) in the nematode Caenorhabditis elegans. We used RNA-seq to investigate how multiple alleles affect gene expression in these mpRILs. We found 1,789 genes differentially expressed between the parental lines. Transgression, expression beyond any of the parental lines in the mpRILs, was found for 7,896 genes. For expression QTL mapping almost 9,000 SNPs were available. By combining these SNPs and the RNA-seq profiles of the mpRILs, we detected almost 6,800 eQTLs. Most trans-eQTLs (63%) co-locate in six newly identified trans-bands. The trans-eQTLs found in previous 2-parental allele eQTL experiments and this study showed some overlap (17.5%- 46.8%), highlighting on the one hand that a large group of genes is affected by polymorphic regulators across populations and conditions, on the other hand it shows that the mpRIL population allows identification of novel gene expression regulatory loci. Taken together, the analysis of our mpRIL population provides a more refined insight into C. elegans complex trait genetics and eQTLs in general, as well as a starting point to further test and develop advanced statistical models for detection of multi-allelic eQTLs and systems genetics studying the genotype-phenotype relationship.

category: genetics


title: Effect of Pre-harvest Calcium Chloride and Salicylic Acid Spray on Morphological and Biochemical Traits of Guava (Psidium guajava)

abstract: Guava is a small, tropical fruit tree grown in various tropical and subtropical regions. Salicylic acid (SA) is a phenolic compound that enhances disease resistance and delays the fruit ripening process. Calcium is an essential cell component that delays ripening, particularly softening of the fruit. The effect of foliar spray of CaCl2, and SA, on the physical and biochemical traits of guava was investigated in the present investigation. The application of CaCl2 2% + SA 2mM was more effective as compared with both when applied alone. The data were recorded on fruit set (%), fruit weight (g), fruit length (cm), fruit diameter (cm), fruit yield (kg), ripening period (days), TSS, acidity, total sugar, ascorbic acid, nitrogen, phosphorus, potassium. CaCl2 2% + SA 2mM was showed better performance in all cases, followed by SA 2mM and CaCl2 2%. Overall, this work determines the influence on guava's essential traits by pre-harvest calcium chloride and salicylic acid.


title: Published Anti-SARS-CoV-2 In Vitro Hits Share Common Mechanisms of Action that Synergize with Antivirals

abstract: The global efforts in the past few months have led to the discovery of around 200 drug repurposing candidates for COVID-19. Although most of them only exhibited moderate anti- SARS-CoV-2 activity, gaining more insights into their mechanisms of action could facilitate a better understanding of infection and the development of therapeutics. Leveraging large-scale drug-induced gene expression profiles, we found 36% of the active compounds regulate genes related to cholesterol homeostasis and microtubule cytoskeleton organization. The expression change upon drug treatment was further experimentally confirmed in human lung primary small airway. Following bioinformatics analysis on COVID-19 patient data revealed that these genes are associated with COVID-19 patient severity. The expression level of these genes also has predicted power on anti-SARS-CoV-2 efficacy in vitro, which led to the discovery of monensin as an inhibitor of SARS-CoV-2 replication in Vero-E6 cells. The final survey of recent drug- combination data indicated that drugs co-targeting cholesterol homeostasis and microtubule cytoskeleton organization processes more likely present a synergistic effect with antivirals. Therefore, potential therapeutics should be centered around combinations of targeting these processes and viral proteins.

category: bioinformatics


title: Mechanical Interplay Between Transcription Elongation and DNA Supercoiling

abstract: Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or "push" between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate via the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the torsional response of supercoiled DNA. Using stochastic simulations, we demonstrate long-range cooperation between co-transcribing RNAPs mediated by DNA supercoiling. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern a key cellular process.


title: DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases


title: Self-organization of Tissue Growth by Interfacial Mechanical Interactions in Multi-layered Systems

abstract: Morphogenesis is a spatially and temporally regulated process involved in various physiological and pathological transformations. In addition to the associated biochemical factors, the physical regulation of morphogenesis has attracted increasing attention. However, the driving force of morphogenesis initiation remains elusive. Here, we show that during the growth of multi-layered tissues, morphogenetic process can be self-organized by the progression of compression gradient stemmed from the interfacial mechanical interactions between layers. In tissues with low fluidity, the compression gradient is progressively strengthened during growth and induces stratification by triggering symmetric-to-asymmetric cell division reorientation at the critical tissue size. In tissues with high fluidity, compression gradient is dynamic and induces cell junction remodelling regulated cell rearrangement leading to 2D in-plane morphogenesis instead of 3D deformation. Morphogenesis can be tuned by manipulating tissue fluidity, cell adhesion forces and mechanical properties to influence the progression of compression gradient during the development of cultured cell sheets and chicken embryos. Together, the dynamics of compression gradient arised from interfacial mechanical interaction provides a conserved mechanism underlying morphogenesis initiation and size control during tissue growth.


title: NKX2-1 controls lung cancer progression by inducing DUSP6 to dampen ERK activity

abstract: Lung cancer remains a leading cause of cancer death, with unclear mechanisms driving the transition to aggressive cancer with poor prognosis. The RAS[->]RAF[->]MEK[->]ERK pathway is hyper-activated in [~]50% of human lung adenocarcinoma (LUAD). An initial activating mutation induces homeostatic feedback mechanisms that limit ERK activity. Additional, undefined genetic hits overcome the feedback, leading to high ERK activity that drives malignant progression. At detection, the majority of LUADs express the homeobox transcription factor NKX2-1, which also limits malignant progression. NKX2-1 constrains LUAD, in part, by maintaining a well-differentiated state with features of pulmonary identity. We asked if loss of NKX2-1 might also contribute to the release of ERK activity that drives tumor progression. Using human tissue samples and cell lines, xenografts, and genetic mouse models, we show that NKX2-1 induces the ERK phosphatase DUSP6. In tumor cells from late-stage LUAD with silenced NKX2-1, re-introduction of NKX2-1 induces DUSP6 and inhibits cell proliferation and migration and tumor growth and metastasis. CRISPR knockout studies show that DUSP6 is necessary for NKX2-1-mediated inhibition of tumor progression in vivo. Further, DUSP6 expression is sufficient to inhibit RAS-driven LUAD. We conclude that NKX2-1 silencing, and thereby DUSP6 downregulation, is a mechanism by which early LUAD can unleash ERK hyperactivation for tumor progression.


title: The M1 aminopeptidase NPEPPS is a novel regulator of cisplatin sensitivity

abstract:
Despite routine use of platinum-based chemotherapeutics in cancer treatment, there remains a need to improve efficacy and patient selection. Multi-omic assessment of human bladder cancer cell lines and their cisplatin resistant derivatives and whole-genome CRISPR screens identified NPEPPS, the puromycin-sensitive aminopeptidase as a novel driver of cisplatin resistance. NPEPPS depletion increased cisplatin import and sensitization of resistant cells in vitro and in vivo. Pharmacologic inhibition of NPEPPS in cells and chemoresistant, patient-derived tumor organoids improved response to cisplatin. NPEPPS was found in complex with volume regulated anion channel (VRAC) subunits LRRC8A and LRRC8D, whose loss is known to enhance resistance to cisplatin. Depletion of LRRC8A the only obligate subunit for normal VRAC function abrogated the effect of NPEPPS-mediated cisplatin import. Our findings describe the first mechanism by which VRACs can be targeted for therapeutic benefit.

HIGHLIGHTSO_LIMulti-omic screening found NPEPPS is a driver of cisplatin resistance in cancer
C_LIO_LINPEPPS is in protein complex with LRRC8A and D, volume regulated anion channel (VRAC) subunits
C_LIO_LILRRC8A and D loss increases cisplatin resistance and reduce its intracellular levels
C_LIO_LINPEPPS regulates cisplatin import and sensitivity through VRACs
C_LIO_LINPEPPS function is inhibited by small molecules offering potential for clinical translation
C_LI


title: NPEPPS regulates intracellular import and sensitivity to cisplatin by interaction with volume regulated anion channels


title: Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants

abstract: Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.


title: Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector

abstract:
The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a novel family of modularly organized T6SS PG degrading effectors with an unprecedented impact in antagonistic bacterial interactions.

One sentence summaryBy modulating local environmental pH through D-Lys secretion, Acinetobacter baumannii enhances the activity of a bifunctional cell wall-targeting T6SS effector, increasing its killing activity against Gram-negative and -positive competitors.


title: Characterization of the Chlamydomonas reinhardtii phycosphere reveals conserved features of the plant microbiota

abstract: Microscopic algae release organic compounds to the region immediately surrounding their cells, known as the phycosphere, constituting a niche for colonization by heterotrophic bacteria. These bacteria take up algal photoassimilates and provide beneficial functions to their host, in a process that resembles the establishment of microbial communities associated with the roots and rhizospheres of land plants. Here, we characterize the microbiota of the model alga Chlamydomonas reinhardtii and reveal extensive taxonomic and functional overlap with the root microbiota of land plants. Reconstitution experiments using synthetic communities derived from C. reinhardtii and Arabidopsis thaliana show that phycosphere and root bacteria assemble into taxonomically equivalent communities on either host. We show that provision of diffusible metabolites is not sufficient for phycosphere community establishment, which additionally requires physical proximity to the host. Our data suggests that the microbiota of photosynthetic organisms, including green algae and flowering plants, assembles according to core ecological principles.


title: A novel, anatomy-similar in vitro model of 3D airway epithelial for anti-coronavirus drug discovery

abstract: SARS-CoV-2 and its induced COVID-19 remains as a global health calamity. Severe symptoms and high mortality, caused by cytokine storm and acute respiratory distress syndrome in the lower respiratory airway, are always associated with elderly individuals and those with comorbidities; whereas mild or moderate COVID-19 patients have limited upper respiratory flu-like symptoms. There is an urgent need to investigate SARS-CoV-2 and other coronaviruses replication and immune responses in human respiratory systems. The human reconstituted airway epithelial air-liquid interface (ALI) models are the most physiologically relevant model for the investigation of coronavirus infection and virus-triggered innate immune signatures. We established ALI models representing both the upper and the lower respiratory airway to characterize the coronavirus infection kinetics, tissue pathophysiology, and innate immune signatures from upper and lower respiratory tract perspective. Our data suggested these in vitro ALI models maintain high physiological relevance with human airway tissues. The coronavirus induced immune response observed in these upper and lower respiratory airway models are similar to what has been reported in COVID-19 patients. The antiviral efficacy results of a few promising anti-coronavirus drugs in these models were consistent with previous reports and could be valuable for the human dose prediction. Taken together, our study demonstrates the importance of 3D airway epithelial ALI model for the understanding of coronavirus pathogenesis and the discovery and development of anti-coronavirus drugs.


title: Generation and mutational analysis of a transgenic mouse model of human SRY

abstract: SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRYs three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and the causes of DSD, and present a versatile and powerful system in which to demonstrate causality of SRY variations in DSD, to functionally study the SRY variation database, and to characterize new pathogenic SRY variations found in DSD.

category: developmental biology


title: Identification and predictability of soil quality factors and indicators from conventional soil and vegetation classifications

abstract: Generally, the physical, chemical and biological attributes of a soil combined with abiotic factors (e.g. climate and topography) drive pedogenesis. However, biological indicators of soil quality play no direct role in traditional soil classification and surveys. To support their inclusion in classification schemes, previous studies have shown that soil type is a key factor determining microbial community composition in arable soils. This suggests that soil type could be used as proxy for soil biological function and vice versa. In this study we assessed the relationship between soil biological indicators with either vegetation cover or soil type. A wide range of soil attributes were measured on soils from across the UK to investigate whether; (1) appropriate soil quality factors (SQFs) and indicators (SQIs) can be identified, (2) soil classification can predict SQIs; (3) which soil quality indicators were more effectively predicted by soil types, and (4) to what extent do soil types and/ or aggregate vegetation classes (AVCs) act as major regulators of SQIs. Factor analysis was used to group 20 soil attributes into six SQFs namely; Soil organic matter, Organic matter humification, Soluble nitrogen, Microbial biomass, Reduced nitrogen and Soil humification index. Of these, Soil organic matter was identified as the most important SQF in the discrimination of both soil types and AVCs. Among the measured soil attributes constituting the Soil organic matter factor were, microbial quotient and bulk density were the most important attributes for the discrimination of both individual soil types and AVCs. The Soil organic matter factor discriminated three soil type groupings and four aggregate vegetation class groupings. Only the Peat soil and Heath and bog AVC were distinctly discriminated from other groups. All other groups overlapped with one another, making it practically impossible to define reference values for each soil type or AVC. We conclude that conventionally classified soil types cannot predict the SQIs (or SQFs), but can be used in conjunction with the conventional soil classifications to characterise the soil types. The two-way ANOVA showed that the AVCs were a better regulator of the SQIs than the soil types and that they (AVCs) presented a significant effect on the soil type differences in the measured soil attributes.

category: ecology


title: Young chicks rely on perceptual grouping to discriminate prime numbers

abstract: Grouping sets of elements into smaller, equal-sized, subsets constitutes a perceptual strategy employed by humans and other animals to enhance cognitive performance. We hypothesized that asymmetrical grouping, a characteristic of prime numbers, could provide visual cues enabling discrimination of prime from non-prime numerosities. Newborn chicks were habituated to even numerosities (as sets of elements presented on a screen), and then tested for their spontaneous choice among a prime and a non-prime odd numerosity. Chicks discriminated and preferred the prime over the composite set of elements. We discuss this result in terms of novelty preference. By employing different contrasts (i.e., 7vs.9, 11vs.9, and 13vs.15) we investigated the limits of such mechanism showing that induced grouping positively affects chicks performance.

category: animal behavior and cognition


title: Kaolinite deposits in the upper Iguacu river, Brazil: formation and mineralogical attributes

abstract: Kaolinite (Kt) is the most studied mineral, being widely used in the ceramic, pharmaceutical and cellulose industries, in addition to being the main soil mineral in the world. Found in different parts of the planet, it differs in genesis and may be formed as a result of local weathering of the rocks, occurring in the silt fraction; and also due to the minerals neogenesis with a predominance of clay-sized particles. The plain of upper Iguacu river has the largest kaolinitic deposit in the south of Brazil and its formation raised doubts if this kaolin was transported or formed in situ due the high organic matter in the alluvial plain. To elucidate the origin of kaolin deposits, we sampled a possible font of the mineral, in the mountains of Serra do Mar and sampled two tubes that reach 4 m depth in the upper Iguacu plain. We performed textural analysis, organic carbon, X-ray diffraction, Kt crystallography in silt and clay fractions, thermal analysis (TDA/TG) to quantify Kt and Gb in the clay fraction and scanning electron microscopy (SEM) with dispersive energy spectroscopy (EDS). The TDA/TG analysis demonstrated that saprolite has 66% of the kaolinite found in the plain. The XRD analysis shows a significant presence of mica (Mc) in all samples of the silt fraction, both in Serra do Mar and in the plain. In SEM/EDS, crystals with planar growth are observed, and the presence of pseudomorphic Mc-Kt in the silt fraction of all analyzed samples, with emphasis on the tubes sample with the crystal having almost twice the size of that observed in the saprolite sample from Serra do Mar, allowing to infer that the Kt of the silt fraction of the wetland soils were formed on site by the diagenesis of mica particles. The results obtained in this work indicate that the kaolinitic material found in the wetland of the upper Iguacu plain is the result of weathering processes in the wetland itself, evidenced by the large pseudomorphs found, even greater than those observed in Serra do Mar.

category: scientific communication and education


title: Non-coding enhancer RNA regulates NPAS4 in the mPFC to control chronic stress-induced anhedonia-like behavior

abstract: Chronic stress can produce reward system deficits (i.e. anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and its required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reduction of pyramidal cell dendritic spine density, revealing a relationship between mPFC dendritic spine changes and anhedonia-like behavior, but not social avoidance behavior. Finally, transcriptomic analysis from the mPFC revealed that NPAS4 influences expression of numerous genes linked to glutamatergic synapses and ribosomal function, as well as many dysregulated genes observed in common neuropsychiatric disorders, including depression. Together our findings reveal an essential role for the activity-regulated transcription factor, NPAS4, in chronic stress-induced mPFC hypofunction and anhedonia.

category: neuroscience


title: NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress- induced anhedonia and dendritic spine loss


title: Induction of Bdnf from promoter I following electroconvulsive seizures contributes to structural plasticity in neurons of the piriform cortex

abstract: The efficacy of electroconvulsive therapy (ECT) as a treatment for psychiatric disorders, including major depressive disorder (MDD) is hypothesized to depend on induction of molecular and cellular events that trigger structural plasticity in neurons. Electroconvulsive seizures (ECS) in animal models can help to inform our understanding of how electroconvulsive therapy (ECT) impacts the brain. ECS induces structural plasticity in neuronal dendrites in many brain regions, including the piriform cortex, a highly epileptogenic region that has also been implicated in depression. ECS-induced structural plasticity is associated with differential expression of unique isoforms encoding the neurotrophin, brain-derived neurotrophic factor (BDNF), but the functional significance of these transcripts in dendritic plasticity is not clear. Here, we demonstrate that different Bdnf isoforms are expressed non-stochastically across neurons of the piriform cortex following ECS. Specifically, cells expressing Bdnf exon 1-containing transcripts show a unique spatial recruitment pattern in response to ECS. We further demonstrate that Bdnf Ex1 expression in these cells is necessary for ECS-induced dendritic spine plasticity.


title: Mature parvalbumin interneuron function in prefrontal cortex requires activity during a postnatal sensitive period

abstract: Sensitive periods in which experience-driven changes in activity persistently shape circuit function are well-described in sensory cortex. Whether comparable periods govern the development of associative cortical areas, like the prefrontal cortex, remains unclear. Here, we focus on the role of activity in the maturation and circuit integration of prefrontal parvalbumin-expressing interneurons, as these cells play an essential role in sensory cortical maturation and develop in lockstep with overall prefrontal circuit function. We found that transiently decreasing prefrontal parvalbumin activity during peripubertal and adolescent development results in persistent impairments in adult functional connectivity, in vivo network function and set-shifting behavior that can be rescued by targeted activation of these interneurons in the adult animal. In contrast, comparable adult inhibition had no lasting effects. These findings identify an activity-dependent sensitive period for prefrontal parvalbumin maturation and highlight how abnormal parvalbumin activity early in life can persistently alter adult circuit function and behavior.


title: Scoping review of the applications of peptide microarrays on the fight against human infections.

abstract:
IntroductionThis scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis.

MethodsElectronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines.

ResultsEighty-six articles from 100 studies were included in the final data charting process. The majority (93%) of the articles were published during 2010-2020 and were mostly from Europe (44%) and North America (34 %). The findings were from the investigation of viral (44%), bacterial (30%), parasitic (25%) and fungal (2%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (78%) were related to mapping B-cell linear epitopes, 10% were on diagnostics, 9% reported on immunosignature characterisation and 6% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling.

ConclusionThe most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.


title: Basement membrane proteins as a substrate for efficient Trypanosoma brucei differentiation in vitro

abstract:
The ability to reproduce the developmental events of trypanosomes that occur in their mammalian host in vitro offers significant potential to assist in understanding of the underlying biology of the process. For example, the transition from bloodstream slender to bloodstream stumpy forms is a quorum-sensing response to the parasite-derived peptidase digestion products of environmental proteins. As an abundant physiological substrate in vivo, we studied the ability of a basement membrane matrix enriched gel (BME) in the culture medium to support differentiation of pleomorphic Trypanosoma brucei to stumpy forms. BME comprises extracellular matrix proteins, which are among the most abundant proteins found in connective tissues in mammals and known substrates of parasite-released peptidases. We previously showed that two of these released peptidases are involved in generating a signal that promotes slender-to-stumpy differentiation. Here, we tested the ability of basement membrane extract to enhance parasite differentiation through its provision of suitable substrates to generate the quorum sensing signal, namely oligopeptides. Our results show that when grown in the presence of BME, T. brucei pleomorphic cells arrest at the G0/1 phase of the cell cycle and express the differentiation marker PAD1, the response being restricted to differentiation-competent parasites. Further, the stumpy forms generated in BME medium are able to efficiently proceed onto the next life cycle stage in vitro, procyclic forms, when incubated with cis-aconitate, further validating the in vitro BME differentiation system. Hence, BME provides a suitable in vitro substrate able to accurately recapitulate physiological parasite differentiation without the use of experimental animals.

Author summaryAfrican trypanosome parasites responsible for human and animal trypanosomiasis, live extracellularly in their mammalian host and exploit environmental information to regulate their virulence and transmissibility. Morphological slender forms that proliferate in the mammalian host transform into the non-proliferative stumpy forms by receiving an extracellular signal generated by the accumulating parasites. Parasites secrete peptidases into the host that degrade extracellular matrix (ECM) proteins. In this study, we evaluated the capacity to generate stumpy forms in vitro by including a basement membrane extract into the culture medium. We show that cells exposed to ECM proteins are able to differentiate efficiently into cell-cycle arrested stumpy forms and that the generated differentiation signal depends on the stumpy induction signalling pathway. This method can be used to generate stumpy forms in vitro, these being suitable for experimental analysis, thereby reducing the use of mice.


title: Nonsterile immunity to cryptosporidiosis in infants is associated with mucosal IgA against the sporozoite and protection from malnutrition

abstract:
We conducted a longitudinal study of cryptosporidiosis from birth to three years of age in an urban slum of Dhaka Bangladesh. Fecal DNA was extracted from monthly surveillance samples and diarrheal stool samples collected from 392 infants from birth to three years. A pan-Cryptosporidium qPCR assay was used to identify sub-clinical and symptomatic cryptosporidiosis. Anthropometric measurements were collected quarterly to assess child nutritional status. 31% (121/392) of children experienced a single and 57% (222/392) multiple infections with Cryptosporidium. Repeat infections had a lower burden of parasites in the stool (Cq slope = -1.85; p<0.0001) and were more likely to be sub-clinical (Chi square test for trend; p=0.01). Repeat infections were associated with the development of growth faltering (Pearson correlation = -0.18; p=0.0004). High levels of fecal IgA antibodies against the Cryptosporidium Cp23 sporozoite protein at one year of life were associated with a delay in reinfection and amelioration of growth faltering through three years of life (HAZ IgA high responders -1.323 {+/-} 0.932 versus HAZ -1.731 {+/-} 0.984 p=0.0001). We concluded that nonsterile immunity to cryptosporidiosis in young children was associated with high levels of mucosal IgA anti-Cp23 and protection from diarrhea and growth faltering.

Authors SummaryCryptosporidium is one of the top causes of diarrhea and growth faltering in Bangladesh infants. We discovered that a prior infection resulted in incomplete immunity that protected from diarrhea and growth faltering but not infection and was associated with mucosal IgA against a sporozoite surface protein Cp23. The most important implication of these findings is that a cryptosporidiosis vaccine may not need to achieve complete protection from infection to have a beneficial impact on child health.


title: NuA4 and SAGA acetyltransferase complexes cooperate for repair of DNA breaks by homologous recombination

abstract:
Chromatin modifying complexes play important yet not fully defined roles in DNA repair processes. The essential NuA4 histone acetyltransferase (HAT) complex is recruited to double-strand break (DSB) sites and spreads along with DNA end resection. As predicted, NuA4 acetylates surrounding nucleosomes upon DSB induction and defects in its activity correlate with altered DNA end resection and Rad51 recombinase recruitment. Importantly, we show that NuA4 is also recruited to the donor sequence during recombination along with increased H4 acetylation, indicating a direct role during strand invasion/D-loop formation after resection. We found that NuA4 cooperates locally with another HAT, the SAGA complex, during DSB repair as their combined action is essential for DNA end resection to occur. This cooperation of NuA4 and SAGA is required for recruitment of ATP-dependent chromatin remodelers, targeted acetylation of repair factors and homologous recombination. Our work reveals a multifaceted and conserved cooperation mechanism between acetyltransferase complexes to allow repair of DNA breaks by homologous recombination.

Key Points-NuA4-dependent acetylation modulates nucleosome dynamics to assist DNA end resection and strand invasion during homologous recombination.
-Combined recruitment and local action of NuA4 and SAGA acetyltransferase complexes is essential for DNA end resection.
-Cooperation between NuA4 and SAGA acetyltransferases is required for homologous recombination in eukaryotes.


title: After migration into blood circulation, hematopoietic stem cells can stably self-renew and maintain bone marrow while being skewed toward myeloid lineage when submitted to serial transplantation.

abstract: Hematopoietic stem cells (HSCs) mainly reside in bone marrow (BM) within niches providing an appropriate environment for their survival and self-renewal. Although, small numbers of HSCs can quit their residing environment to migrate into blood circulation and re-engraft elsewhere in BM. Mobilizing agents such as granulocyte colony stimulating factor (G-CSF) can amplify this process by inducing massive HSC mobilization into blood circulation. This method is widely used in clinics to treat hematological disorders. However, in physiological conditions, the properties of HSCs after migration (called migratory HSCs) remain incompletely characterized. In this study, we investigated the capacity of migratory HSCs to self-renew, reconstitute and maintain BM. We show that after migration, HSCs can stably self-renew and maintain BM in homeostasis. However, while stably repopulating BM of irradiated recipients, migratory HSCs show a defect in lymphoid lineage reconstitution when subjected to serial transplantations. Our findings provide interesting knowledge on HSC properties after migration, which may benefits therapeutic research on HSC-based therapies to treat hematological disorders.

category: cell biology


title: The parathyroid hormone-dependent activation of osteoblasts enhances hematopoietic stem cell migration and reduces their engraftment abilities

abstract: Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in HSC niches ensuring their maintenance. The HSC niche is made up of perivascular and trabecular cells including osteoblasts whose role on HSCs remains to be clearly defined. Increased numbers of osteoblasts have been observed in the CL2 transgenic mouse expressing a constitutively activated form of the parathyroid hormone (PTH)/PTH-related peptide receptor. This mouse model mimicking PTH anabolic effect has also been described to exhibit increased numbers of the BM stem/progenitor population. Furthermore, PTH is known to induce BM stem/progenitor cell migration into blood circulation. However PTH role on long-term repopulating HSCs (LT-HSCs) is incompletely known. Here we show that CL2 BM contains a regular proportion of LT-HSCs, suggesting that osteoblasts may not be a determinant of LT-HSC numbers but act mainly on more mature progenitors. We further show increased LT-HSC migration in CL2 mice correlated with higher granulocyte colony-stimulating factor (G-CSF) serum levels, supporting the idea that PTH can enhance the migration of LT-HSCs. Finally, we found a defect in the ability of CL2 BM HSCs to reconstitute irradiated BM suggesting that PTH activation of osteoblasts negatively influences abilities of HSC population to engraft and reconstitute irradiated BM. In summary, our study highlights new insights into the role of the PTH-dependent activation of osteoblasts on LT-HSC migration and their BM repopulation abilities. Our findings will be useful to improve treatments on hematological disorders, especially therapies involving HSC harvest and transplantation.


title: Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation

abstract:
BackgroundThe NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly.

MethodsTo explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts.

ResultsPKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1{beta} production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly.

ConclusionAlthough PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.


title: Deep learning image analysis for in vivo shortwave infrared imaging

abstract: Shortwave infrared window (SWIR: 1000-1700 nm) represents a major improvement compared to the NIR-I region (700-900 nm) in terms of temporal and spatial resolutions in depths down to 4 mm. SWIR is a fast and cheap alternative to more precise methods such as X-ray and opto-acoustic imaging. Main obstacles in SWIR imaging are the noise and scattering from tissues and skin that reduce the precision of the method. We demonstrate that the combination of SWIR in vivo imaging in the NIRIIb region (1500-1700 nm) with advanced deep learning image analysis allows to overcome these obstacles and making a large step forward to high resolution imaging: it allows to precisely segment vessels from tissues and noise, provides morphological structure of the vessels network, with learned pseudo-3D shape, their relative position, dynamic information of blood vascularization in depth in small animals and distinguish the vessels types: artieries and veins. For demonstration we use neural network IterNet that exploits structural redundancy of the blood vessels, which provides a useful analysis tool for raw SWIR images.


title: Deep learning: step forward to high-resolution in vivo shortwave infrared imaging


title: Direct molecular evidence for an ancient, conserved developmental toolkit controlling post-transcriptional gene regulation in land plants

abstract: RNA interference (RNAi) plays important roles in organism development through post-transcriptional regulation of specific target mRNAs. Target specificity is largely controlled by base-pair complementarity between micro-RNA (miRNA) regulatory elements and short regions of the target mRNA. The pattern of miRNA production in a cell interacts with the cells mRNA transcriptome to generate a specific network of post-transcriptional regulation that can play critical roles in cellular metabolism, differentiation, tissue/organ development and developmental timing. In plants, miRNA production is orchestrated in the nucleus by a suite of proteins that control transcription of the pri-miRNA gene, post-transcriptional processing and nuclear export of the mature miRNA. In the model plant, Arabidopsis thaliana, post-transcriptional processing of miRNAs is controlled by a pair of physically-interacting proteins, HYL1 and DCL1. However, the evolutionary history of the HYL1-DCL1 interaction is unknown, as is its structural basis. Here we use ancestral sequence reconstruction and functional characterization of ancestral HYL1 in vitro and in vivo to better understand the origin and evolution of the HYL1-DCL1 interaction and its impact on miRNA production and plant development. We found the ancestral plant HYL1 evolved high affinity for both double-stranded RNA (dsRNA) and its DCL1 partner very early in plant evolutionary history, before the divergence of mosses from seed plants (~500 Ma), and these high-affinity interactions remained largely conserved throughout plant evolutionary history. Structural modeling and molecular binding experiments suggest that the second of two double-stranded RNA-binding motifs (DSRMs) in HYL1 may interact tightly with the first of two C-terminal DCL1 DSRMs to mediate the HYL1-DCL1 physical interaction necessary for efficient miRNA production. Transgenic expression of the nearly 200 Ma-old ancestral flowering-plant HYL1 in A. thaliana was sufficient to rescue many key aspects of plant development disrupted by HYL1- knockout and restored near-native miRNA production, suggesting that the functional partnership of HYL1-DCL1 originated very early in and was strongly conserved throughout the evolutionary history of terrestrial plants. Overall, our results are consistent with a model in which miRNA-based gene regulation evolved as part of a conserved plant  developmental toolkit; its role in generating developmental novelty is probably related to the relatively rapid evolution of miRNA genes.

category: evolutionary biology


title: Large-scale placenta DNA methylation mega-analysis reveals fetal sex-specific differentially methylated CpG sites and regions

abstract: Although male-female differences in placental structure and function have been observed, little is understood about their molecular underpinnings. Here, we present a mega-analysis of 14 publicly available placenta DNA methylation (DNAm) microarray datasets to identify individual CpGs and regions associated with fetal sex. In the discovery dataset of placentas from full term pregnancies (N = 532 samples), 5,212 CpGs met genome-wide significance (p < 1E-8) and were enriched in pathways such as keratinization (FDR p-value = 7.37E-14), chemokine activity (FDR p-value = 1.56E-2), and eosinophil migration (FDR p-value = 1.83E-2). Nine differentially methylated regions were identified (fwerArea < 0.1) including a region in the promoter of ZNF300 that showed consistent differential DNAm in samples from earlier timepoints in pregnancy and appeared to be driven predominately by effects in the trophoblast cell type. We describe the largest study of fetal sex differences in placenta DNAm performed to date, revealing genes and pathways characterizing sex-specific placenta function and health outcomes later in life.

category: genomics


title: scCorr: A graph-based k-partitioning approach for single-cell gene-gene correlation analysis

abstract: An important challenge in single-cell RNA-sequencing analysis is the abundance of zero values, which results in biased estimation of gene-gene correlations for downstream analyses. Here, we present a novel graph-based k-partitioning method by merging "homology" cells to reduce the number of zero values. Our method is robust and reliable for the detection of correlated gene pairs, which is fundamental to network construction, gene-gene interaction, and cellular -omic analyses.


title: Haplotype-aware variant calling enables high accuracy in nanopore long-reads using deep neural networks

abstract: Long-read sequencing has the potential to transform variant detection by reaching currently difficult-to-map regions and routinely linking together adjacent variations to enable read based phasing. Third-generation nanopore sequence data has demonstrated a long read length, but current interpretation methods for its novel pore-based signal have unique error profiles, making accurate analysis challenging. Here, we introduce a haplotype-aware variant calling pipeline PEPPER-Margin-DeepVariant that produces state-of-the-art variant calling results with nanopore data. We show that our nanopore-based method outperforms the short-read-based single nucleotide variant identification method at the whole genome-scale and produces high-quality single nucleotide variants in segmental duplications and low-mappability regions where short-read based genotyping fails. We show that our pipeline can provide highly-contiguous phase blocks across the genome with nanopore reads, contiguously spanning between 85% to 92% of annotated genes across six samples. We also extend PEPPER-Margin-DeepVariant to PacBio HiFi data, providing an efficient solution with superior performance than the current WhatsHap-DeepVariant standard. Finally, we demonstrate de novo assembly polishing methods that use nanopore and PacBio HiFi reads to produce diploid assemblies with high accuracy (Q35+ nanopore-polished and Q40+ PacBio-HiFi-polished).


title: Engineered SARS-CoV-2 receptor binding domain improves immunogenicity in mice and elicits protective immunity in hamsters

abstract: Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs).1 Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access.2 Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing costs.3 These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples.4-6 Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2.7,8 Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.


title: In situ community dynamics influences the temperature- and light- dependent succession of seasonal phytoplankton

abstract: Temperature and light play a crucial role in regulating phytoplankton blooms in the Ocean. To assess the importance of these two parameters experimentally, microcosms were conducted on seven picoplankton communities (<3 m) sampled in December, March, June and September 2015 and 2016 in the North Western Mediterranean Sea. Each community was exposed to 4 realistic seasonal conditions (December, March, June and September). Metabarcoding was used to investigate the eukaryotic diversity in the 56 microcosms experiments in parallel to high-frequency monitoring of environmental diversity in the sea. The three major lineages identified were the Stramenopiles, Alveolata and Archaeplastida. Overall, the five-day incubations were not sufficient to reshape the initial microbial communities completely. The microcosm outcome was strongly influenced by the dynamics of phytoplankton starting communities. In pre-bloom conditions, phytoplanktonic species were the most sensitive to temperature and light conditions. During a bloom, species belonging to diatoms or Chlorodendrophyceae usually did not respond to light and temperature in microcosms and continued to bloom independently of the applied seasonal condition. Together, these results suggest that light and temperature seasonal conditions play a crucial role in regulating phytoplankton dynamics in pre-bloom conditions and biotic interactions may be preponderant in bloom and post-bloom conditions.


title: Intra-species transcriptional profiling reveals key regulators of Candida albicans pathogenic traits

abstract:
The human commensal and opportunistic fungal pathogen Candida albicans displays extensive genetic and phenotypic variation across clinical isolates. Here, we performed RNA sequencing on 21 well-characterized isolates to examine how genetic variation contributes to gene expression differences, and to link these differences to phenotypic traits. C. albicans adapts primarily through clonal evolution and yet hierarchical clustering of gene expression profiles in this set of isolates did not reproduce their phylogenetic relationship. Strikingly, strain-specific gene expression was prevalent in some strain backgrounds. Association of gene expression with phenotypic data by differential analysis, linear correlation, and assembly of gene networks connected both previously characterized and novel genes with 23 C. albicans traits. Construction of de novo gene modules produced a gene atlas incorporating 67% of C. albicans genes and revealed correlations between expression modules and important phenotypes such as systemic virulence. Furthermore, targeted investigation of two modules that have novel roles in growth and filamentation supported our bioinformatic predictions. Together, these studies reveal widespread transcriptional variation across C. albicans isolates and identify genetic and epigenetic links to phenotypic variation based on co-expression network analysis.

ImportanceInfectious fungal species are often treated uniformly despite clear evidence of genotypic and phenotypic heterogeneity being widespread across strains. Identifying the genetic basis for this phenotypic diversity is extremely challenging because of the tens or hundreds of thousands of variants that may distinguish two strains. Here we use transcriptional profiling to determine differences in gene expression that can be linked to phenotypic variation among a set of 21 Candida albicans isolates. Analysis of this transcriptional dataset uncovered clear tends in gene expression characteristics for this species and new genes and pathways that associated with variation in pathogenic processes. Direct investigation confirmed functional predictions for a number of new regulators associated with growth and filamentation, demonstrating the utility of these approaches in linking genes to important phenotypes.


title: Sector search strategies for odor trail tracking

abstract: Terrestrial animals such as ants, mice and dogs often use surface-bound scent trails to establish navigation routes or to find food and mates, yet their tracking strategies are poorly understood. Tracking behavior features zig-zagging paths with animals often staying in close contact with the trail. Upon sustained loss of contact, animals execute a characteristic sequence of sweeping "casts" - wide oscillations with increasing amplitude. Here, we provide a unified description of trail-tracking behavior by introducing an optimization framework where animals search in the angular sector defined by their estimate of the trails heading and its uncertainty. In silico experiments using reinforcement learning based on this hypothesis recapitulate experimentally observed tracking patterns. We show that search geometry imposes limits on the tracking speed, and quantify its dependence on trail statistics and memory of past contacts. By formulating trail-tracking as a Bellman-type sequential optimization problem, we quantify the basic geometric elements of optimal sector search strategy, effectively explaining why and when casting is necessary. We propose a set of experiments to infer how tracking animals acquire, integrate and respond to past information on the tracked trail. More generally, we define navigational strategies relevant for animals and bio-mimetic robots, and formulate trail-tracking as a novel behavioral paradigm for learning, memory and planning.


title: SPOT: a web-tool enabling Swift Profiling Of Transcriptomes

abstract: The increasing number of single cell and bulk RNAseq data sets describing complex gene expression profiles in different organisms, organs or cell types calls for an intuitive tool allowing rapid comparative analysis. Here we present Swift Profiling Of Transcriptomes (SPOT) as a web tool that allows not only differential expression analysis but also fast ranking of genes fitting transcription profiles of interest. Based on a heuristic approach the spot algorithm ranks the genes according to their proximity to the user-defined gene expression profile of interest. The best hits are visualized as a table, bar chart or dot plot and can be exported as an Excel file. While the tool is generally applicable, we tested it on RNAseq data from malaria parasites that undergo multiple stage transformations during their complex life cycle as well as on data from multiple human organs during development and cell lines infected by the SARS-CoV-2 virus. SPOT should enable non-bioinformaticians to easily analyse their own and any available dataset.


title: A machine learning approach for detecting vicarious trial and error behaviors

abstract: Vicarious trial and error behaviors (VTEs) indicate periods of indecision during decision-making, and have been proposed as a behavioral marker of deliberation. In order to understand the neural underpinnings of these putative bridges between behavior and neural dynamics, researchers need the ability to readily distinguish VTEs from non-VTEs. Here we utilize a small set of trajectory-based features and standard machine learning classifiers to identify VTEs from non-VTEs for rats performing a spatial delayed alternation task (SDA) on an elevated plus maze. We also show that previously reported features of the hippocampal field potential oscillation can be used in the same types of classifiers to separate VTEs from non-VTEs with above chance performance. However, we caution that the modest classifier success using hippocampal population dynamics is not sufficient for identifying trials where VTEs occur, and show that combining oscillation-based features with trajectory-based features degrades classifier performance compared to trajectory-based features alone. Overall, we propose a standard set of features useful for trajectory-based VTE classification and support previous suggestions that VTEs are supported by a network including, but likely extending beyond, the hippocampus.