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Results for “molecular biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Molecular and cellular pathology of monogenic Alzheimer's disease at single cell resolution

Cell and molecular biology analyses of sporadic Alzheimers disease brain are confounded by clinical variability, ageing and genetic heterogeneity. Therefore, we used single-nucleus RNA sequencing to characterize cell composition and gene expression in the cerebral cortex in early-onset, monogenic Alzheimers disease. Constructing a cellular atlas of frontal cortex from 8 monogenic AD individuals and 8 matched controls, provided insights into which neurons degenerate in AD and responses of different cell types to AD at the cellular and systems level. Such responses are a combination of positively adaptive and deleterious changes, including large-scale changes in synaptic transmission and marked metabolic reprogramming in neurons. The nature and scale of the transcriptional changes in AD emphasizes the global impact of the disease across all brain cell types. One Sentence SummaryAlzheimers disease brain atlas provides insights into disease mechanisms

neuroscience↗

bioRxiv2020

Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation

The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into Petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB-deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the Petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, as-yet-unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements. ImportanceThis first molecular biological study of archaeal immersed liquid biofilms advances our basic-biological understanding of the model archaeon Haloferax volcanii. Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.

microbiology↗

bioRxiv2020

Integrative Vectors for Regulated Expression of SARS-CoV-2 Proteins Implicated in RNA Metabolism

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His8, the C-terminus with His8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In TREx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

cell biology↗

bioRxiv2020

Modulation of proteasome activity by curcumin and didemethylcurcumin

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways and known to modulate PKC activities. The therapeutic significance of curcumin is often considered to reside in its anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. However, recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. In this study, we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin), CUIII modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in relative proteasome activity increase at nanomolar concentrations. Also, curcumin was more effective than CUIII in relative proteasome activity inhibition at micromolar concentration. The docking study was conducted on the 20S proteasome catalytic subunit. Estimated Kd values for curcumin and didemethylcurcumin are 0.0054M and 1.3167M, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compare to CUIII. The small size of CUIII makes its dock to the narrow cavity of the active site, but the binding interaction is not strong compare to curcumin. This study suggests that curcumin and its didemethyl derivative can be used to modulate proteasome activity. This communication implicates the reason why curcumin and its didemethyl derivative can be used to two different disease mechanisms, neurodegeneration, and cancer.

neuroscience↗

bioRxiv2020

Selecting Student-Authored Questions for Summative Assessments

Production of high-quality multiple-choice questions (MCQs) for both formative and summative assessment is a time-consuming task requiring great skill, creativity, and insight. The transition to online examinations, with the concomitant exposure of previously tried-and-tested MCQs, exacerbates the challenges of question production, and highlights the need for innovative solutions. Several groups have shown that it is practical to leverage the student cohort to produce a very large number of syllabus-aligned MCQs for study banks. Although student-generated questions are well suited for formative feedback and practice activities, they are generally not thought to be suitable for high-stakes assessments. In this study, we aimed to demonstrate that training can be provided to students in a scalable fashion to generate questions of similar quality to those produced by experts, and that identification of suitable questions can be achieved with minimal academic review and editing. Biochemistry and Molecular Biology students were assigned a series of activities designed to coach them in the art of writing and critiquing MCQs. This training resulted in the production of over one thousand MCQs that were then gauged for potential by either expert academic judgement, or via a data-driven approach in which the questions were trialled objectively in a low-stakes test. Questions selected by either method were then deployed in a high-stakes in-semester assessment alongside questions from two academically authored sources: textbook-derived MCQs, and past paper questions. A total of 120 MCQs from these four sources were deployed in assessments attempted by over 600 students. Each question was subjected to rigorous performance analysis, including the calculation of standard metrics from classical test theory and more sophisticated Item Response Theory (IRT) measures. The results showed that MCQs authored by students and selected at low cost performed as well as questions authored by academics, illustrating the potential of this strategy for the efficient creation of large numbers of high quality MCQs for summative assessment.

scientific communication and education↗

bioRxiv2020

Knock-in of labeled proteins into 5'UTR enables highly efficient generation of stable cell lines

Stable cell lines and animal models expressing tagged proteins are important tools for studying behaviors of cells and molecules. Several molecular biological technologies have been applied with varying degrees of success and efficiencies to establish cell lines expressing tagged proteins. Here we applied CRISPR/Cas9 for the knock-in of tagged proteins into the 5UTR of the endogenous gene loci. With this 5UTR-targeting knock-in strategy, stable cell lines expressing Arl13b-Venus, Reep6-HA, and EGFP-alpha-tubulin were established with high knock-in efficiencies ranging from 50 to 80%. The localization of the knock-in proteins were identical to that of the endogenous proteins in wild-type cells and showed homogenous expression. Moreover, the expression of knock-in EGFP-alpha-tubulin from the endogenous promoter was stable over long-term culture. We further demonstrated that the fluorescent signals were enough for a long time time-lapse imaging. The fluorescent signals were distinctly visible during the whole duration of the time-lapse imaging and showed specific subcellular localizations. Altogether, our strategy demonstrates that 5UTR is a hotspot for targeted insertion of gene sequences and allows the stable expression of tagged proteins from endogenous loci in mammalian cells.

cell biology↗

bioRxiv2020

Interpretation of exercise-induced changes in human skeletal muscle mRNA expression depends on the timing of the post-exercise biopsies

AimExercise elicits a range of adaptive responses in skeletal muscle that include changes in mRNA expression. To better understand the health benefits of exercise training, it is essential to investigate the underlying molecular mechanisms of skeletal muscle adaptations to exercise. However, most studies have assessed the molecular events at a few convenient time points within a short time frame post exercise, and the variations of gene expression kinetics have not been addressed systematically. MethodMuscle biopsies were collected from nine participants at baseline and six time points (0, 3, 9, 24, 48, and 72 h) following a session of high-intensity interval exercise. We assessed the mRNA content of 23 gene isoforms from the muscle samples. ResultThe temporal patterns of target gene expression were highly variable and the mRNA contents detected were largely dependent on the muscle sample timing. The maximal levels of mRNA content of all tested target genes were observed between 3 to 48 h post exercise. ConclusionOur findings highlight a critical gap in knowledge regarding the molecular response to exercise, where the use of a few time points within a short period after exercise has led to an incomplete understanding of the molecular responses to exercise. The timing of muscle sampling for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.

physiology↗

bioRxiv2020

Distinct molecular targets for macrocyclic lactone and isoxazoline insecticides in the human louse: new prospects for the treatment of pediculosis

BackgroundControl of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of resistant parasites to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanism of action remain largely unknown. The macrocyclic lactone, ivermectin is active against a broad range of insects including human lice. Isoxazolines are a recent new chemical class targeting the {gamma}-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and exhibiting a strong insecticidal potential. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the novel ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Methods and findingsUsing toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and glutamate-gated chloride channel subunits (Phh-GluCl) were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 6.4 {micro}M and 9.3 {micro}M. Importantly, ivermectin displayed a super agonist action on Phh-Glucl whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value 0.5 {micro}M, whereas it had no effect on Phh-GluCl. ConclusionsWe report here for the first time the pediculicidal potential of isoxazolines and reveal the distinct mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize the clear repurposing future of the isoxazoline drug class to provide new pediculicidal agents to tackle resistant-louse infestations in humans. Autorship summaryO_ST_ABSWhy was this study done?C_ST_ABSHuman cosmopolitan lice are responsible for pediculosis, which represent a significant public health concern. Resistant lice against insecticides and lack of safety of the treatments for human and environment is a growing issue worldwide. Here we investigated the efficacy on lice of the classical macrocyclic lactone drug, ivermectin, and the novel isoxazoline drug, lotilaner. This study was done to decipher their mode of action at the molecular and funtional levels in order to propose new strategies to control lice infestation. What did the researchers do and find?Our bioassay results indicate that ivermectin and lotilaner were potent at killing human adult lice, with lotilaner showing a higher efficacy than ivermectin. Furthermore, we identified and pharmacologically characterized the first glutamate- and GABA-gated chloride channels ever described in human lice yet. Mechanistically, our molecular biology and electrophysiology findings demonstrate that ivermectin acted preferentially at glutamate channels while lotilaner specifically targeted GABA channels. What do these findings mean?These results provide new insights in the understanding of the insecticide mode of action and highlight the isoxazolines as a new drug-repurposing opportunity for pest control strategies.

pharmacology and toxicology↗

bioRxiv2020

Generation of tonsil organoids as an ex vivo model for SARS-CoV-2 infection

Palatine tonsil (hereinafter referred to as "tonsil") plays role in the immune systems first line of defense against foreign pathogens. Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a worldwide pandemic since the infection was first reported in China in December 2019. The aim of this study was to establish tonsil epithelial cell-derived organoids and to examine their feasibility as an ex vivo model for SARS-CoV-2 infection. Using an optimized protocol, we achieved 3D tonsil organoid culture from human tonsil tissue that reflects the distinctive characteristics of the tonsil epithelium, such as its cellular composition, histologic properties, and molecular biological features. Notably, we verified that SARS-CoV-2 can infect tonsil organoids with a robust replication efficiency. Furthermore, treatment with remdesivir, an antiviral agent, effectively protected them from viral infection. Therefore, tonsil organoids could be available for investigation of SARS-CoV-2 infection-mediated pathology and for preclinical screening of novel antiviral drug candidates. One-sentence SummaryThis study established tonsil epithelial cell-derived organoids and demonstrated their feasibility as an ex vivo model for SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

Stool methanogens in intestine mammal species.

Methanogens are being members of anaerobe microbiota of the digestive tract of both human and mammals. However, the sources, modes of acquisition and dynamics of digestive tract methanogens remain poorly investigated. In this study, we aimed to expand the spectrum of animals which could be sources of methanogens for human, by exploring methanogen carriage in animals in contact with the general population or with some restricted populations; comparing the repertoire of animal methanogens with the one of human methanogens in order to question methanogens as zoonotic microorganisms. We used RT-PCR, PCR-sequencing and multispacer sequence typing to investigate the presence of methanogens in 407 fecal specimens collected from nine different mammalian species. We detected by RT-PCR, the presence of methanogen DNA in all mammals here investigated and none of the negative controls. We obtained by sequencing, seven different species of methanogens, of which three (Methanobrevibacter smithii, Methanobrevibacter millerae and Methanomassiliicoccus luminyensis) are known to be part of the methanogens present in the human digestive tract. We obtained 24 M. smithii by PCR-sequencing including 12/24 (50%) in pigs, 6/24 (25%) in dogs, 4/24 (16.66%) in cats, and 1/24 (4.16%) in both sheep and horses. Genotyping these 24 M. smithii revealed five different genotypes, all know in humans. Our results are fairly representative of the methanogen community present in the digestive tract of certain animals domesticated by humans and other future studies must be done to try to cultivate methanogens here detected by molecular biology to better understand the dynamics of methanogens in animals and also the likely acquisition of methanogens in humans through direct contact with these animals or through consumption of the meat and/or milk of certain animals, in particular cows.

microbiology↗

bioRxiv2020

Experimental pieces of evidence for Mycobacterium ulcerans dormancy.

BackgroundWhether Mycobacterium ulcerans, the etiological agent of the neglected Buruli ulcer in numerous tropical countries, would exist in a dormant state as reported for closely related Mycobacterium species, is not established. MethodologySix M. ulcerans strains were exposed to a progressive depletion in oxygen for two months, using a previously described Wayne model of dormancy; and further examined by microscopy using DDD staining, microcalorimetry and subculture in the presence of dead and replicative M. ulcerans as controls. Principal Findings/ConclusionsM. ulcerans CU001 strain died during the progressive oxygen depletion and four of five remaining strains exhibited Nile Red-stained intracellular lipid droplets after DDD staining and a 14-20-day regrowth when exposed to ambient air, diagnosing dormancy. A fifth M. ulcerans 19423 strain stained negative in DDD and slowly regrew in 27 days. Three tested M. ulcerans strains yielded microcalorimetric pattern similar to that of the negative (dead) homologous controls, differing from that of the homologous positive (replicative) controls. The relevance of these experimental observations, suggesting a previously unreported dormancy state of M. ulcerans, needs to be investigated in the natural ecological niches where M. ulcerans thrive and in Buruli ulcer lesions. Author summaryMycobacterium ulcerans is an environmental opportunistic pathogen of mammals and humans, causing a subcutaneous necrotizing infection named Buruli ulcer. Molecular detection of M. ulcerans DNA revealed different ecological niches where M. ulcerans may thrive, but the molecular biology approach does not catch the physiological state of M. ulcerans in these different ecological niches. Thus, the reservoir and the mode of transmission of M. ulcerans remain elusive. Here, we investigated experimental dormancy of M. ulcerans by using a previously described Wayne model of dormancy coupled with microscopy using DDD staining, microcalorimetry and subculture. Our findings demonstrate for the first time that some M. ulcerans strains exhibit a physiological state of dormancy; potentially limiting isolation and culture of M. ulcerans from environmental niches.

microbiology↗

bioRxiv2020

Identification of genetic factors controlling phosphorus utilization efficiency in wheat by genome-wide association study with principal component analysis

Despite the economic importance of P utilization efficiency, information on genetic factors underlying this trait remains elusive. To address that, we performed a genome-wide association study in a spring wheat diversity panel ranging from landraces to elite varieties. We evaluated the phenotype variation for P utilization efficiency in controlled conditions and genotype variation using wheat 90K SNP array. Phenotype variables were transformed into a smaller set of uncorrelated principal components that captured the most important variation data. We identified two significant loci associated with both P utilization efficiency and the 1st principal component on chromosomes 3A and 4A: qPE1-3A and qPE2-4A. Annotation of genes at these loci revealed 53 wheat genes, among which 6 were identified in significantly enriched pathways. The expression pattern of these 6 genes indicated that TraesCS4A02G481800, involved in pyruvate metabolism and TCA cycle, had a significantly higher expression in the P efficient variety under limited P conditions. Further characterization of these loci and candidate genes can help stimulate P utilization efficiency in wheat. KEY MESSAGEWe report two new loci for P utilization efficiency on chromosomes 3A and 4A of wheat. The prioritized candidate genes at these loci can be investigated by molecular biology techniques to improve P efficiency in wheat and grass relatives.

genomics↗

bioRxiv2020

Hopeful monsters: Unintended sequencing of famously malformed mite mitochondrial tRNAs reveals widespread expression and processing of sense-antisense pairs

Although tRNA structure is one of the most conserved and recognizable shapes in molecular biology, aberrant tRNAs are frequently found in the mitochondrial genomes of metazoans. The structure of several mitochondrial tRNAs is so degenerate that doubts have been raised about their expression and function. Mites from the arachnid superorder Acariformes are predicted to have some of the shortest mitochondrial tRNAs, with apparent base mismatches in acceptor stems and a complete loss of cloverleaf-life shape. While performing mitochondrial isolations and recently developed RNA-seq methods to capture mature, CCA-tailed tRNAs in plant tissue, we inadvertently sequenced the mitochondrial tRNAs from a common plant pest, the acariform mite Tetranychus urticae, to a high enough coverage to detect all previously annotated T. urticae tRNA regions. The results not only confirm expression, CCA-tailing and post-transcriptional base modification of these highly divergent tRNAs, but also revealed widespread sense and antisense expression of tRNA genes in the T. urticae mitochondrial genome. Mirrored expression of mitochondrial tRNA genes has been previously hypothesized but not demonstrated to be extensive in any system. We discuss the functional roles that these divergent tRNAs could have as both decoding molecules in translation and processing signals in transcript maturation pathways for other genes, as well as how the surprising finding of sense-antisense tRNA pairs adds another dimension to the bizarre tRNA biology of mitochondrial genomes.

evolutionary biology↗

bioRxiv2020

Polee: RNA-Seq analysis using approximate likelihood

The analysis of mRNA transcript abundance with RNA-Seq is a central tool in molecular biology research, but often analyses fail to account for the uncertainty in these estimates, which can be significant, especially when trying to disentangle isoforms or duplicated genes. Preserving un-certainty necessitates a full probabilistic model of the all the sequencing reads, which quickly becomes intractable, as experiments can consist of billions of reads. To overcome these limitations, we propose a new method of approximating the likelihood function of a sparse mixture model, using a technique we call the Polya tree transformation. We demonstrate that substituting this approximation for the real thing achieves most of the benefits with a fraction of the computational costs, leading to more accurate detection of differential transcript expression. AvailabilityThe method is implemented in a Julia package available from https://github.com/dcjones/polee Contactdcjones@cs.washington.edu

bioinformatics↗

bioRxiv2020

Proximity-dependent proteomics reveals extensive interactions of Protocadherin-19 with regulators of Rho GTPases and the microtubule cytoskeleton

Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.

neuroscience↗

bioRxiv2020

Actin cytoskeletal and focal adhesion organisation in U2OS cells on polymer nanostructures

Cells in their natural environment are embedded in a complex surrounding consisting of biochemical and biomechanical cues directing cell properties and cell behaviour. Nonetheless, in vitro cell studies are typically performed on flat surfaces, with clear differences from the more complex situation cells experience in vivo. To increase the physiological relevance of these studies, a number of advanced cellular substrates for in vitro studies have been applied. One of these approaches include flat surfaces decorated with vertically aligned nanostructures. In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays. This change is analogous to similar responses for cells seeded on soft substrates. Overall, we show that the combination of high throughput nanofabrication, advanced optical microscopy, molecular biology tools to visualise cellular processes and data analysis can be used to investigate how cells interact with nanostructured surfaces and will in the future help to create culture substrates that induce particular cell function.

biophysics↗

bioRxiv2020

A novel Hsp90 phospho-switch modulates virulence in the major human fungal pathogen Candida albicans

The ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. Hsp90 also regulates cellular morphogenesis, drug resistance, and virulence in human pathogenic fungi, which kill more than 1.6 million patients each year worldwide. Invasive fungal infections are difficult to treat due to the lack of effective antifungal therapies, resulting in mortality rates of up to 95%. As a key regulator of fungal virulence, Hsp90 is an attractive therapeutic target. However, fungal and animal homologs are highly conserved, impeding fungal-specific targeting. Thus, understanding the factors that regulate Hsp90 could provide an alternative strategy aimed at exclusively targeting this regulator of fungal virulence. Here, we demonstrate how CK2-mediated phosphorylation of two Hsp90 residues modulates virulence in a major fungal pathogen of humans, Candida albicans. We combined proteomics, molecular evolution and structural modelling with molecular biology to identify and characterize two Hsp90 phosphorylation sites. Phosphorylation negatively affects thermal stress response, morphogenesis, drug susceptibility and fungal virulence. Our results provide the first record of specific Hsp90 phosphorylation sites acting as modulators of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitation as antifungal drug targets.

microbiology↗

bioRxiv2020

Potent mouse monoclonal antibodies that block SARS-CoV-2 infection

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a global pandemic since its first outbreak in the winter of 2019. An extensive investigation of SARS-CoV-2 is critical for disease control. Various recombinant monoclonal antibodies of human origin that neutralize SARS-CoV-2 infection have been isolated from convalescent patients and will be applied as therapies and prophylaxis. However, the need for dedicated monoclonal antibodies in molecular pathology research is not fully addressed. Here, we produced mouse anti-SARS-CoV-2 spike monoclonal antibodies that exhibit not only robust performance in immunoassays including western blotting, ELISA, immunofluorescence, and immunoprecipitation, but also neutralizing activity against SARS-CoV-2 infection in vitro. Our monoclonal antibodies are of mouse origin, making them compatible with the experimental immunoassay setups commonly used in basic molecular biology research laboratories, and large-scale production and easy distribution are guaranteed by conventional mouse hybridoma technology.

microbiology↗