title: The evolution and biological correlates of hand preferences in anthropoid primates

abstract: The evolution of human right-handedness has been intensively debated for decades. Manual lateralization patterns in non-human primates have the potential to elucidate evolutionary determinants of human handedness. However, restricted species samples and inconsistent methodologies are limiting comparative phylogenetic studies. By combining original data with published literature reports, we assembled data on hand preferences for standardized object manipulation in 1,806 individuals from 38 species of anthropoid primates, including monkeys, apes, and humans. Based on that, we employ quantitative phylogenetic methods to test prevalent hypotheses on the roles of ecology, brain size and tool use in primate handedness evolution. We confirm that human right-handedness represents an unparalleled extreme among anthropoids and found taxa displaying significant population-level handedness to be rare. Species-level direction of manual lateralization was largely uniform among non-human primates and neither correlated with phylogeny nor with any of the selected biological predictors. In contrast, we recovered highly variable patterns of hand preference strength, which show signatures of both ecology and phylogeny. In particular, terrestrial primates tend to display weaker hand preferences than arboreal species. These results challenge popular ideas on primate handedness evolution, especially the postural origins hypothesis. Furthermore, they point to a potential adaptive benefit of disparate lateralization strength in primates, a measure of hand preference that has often been overlooked in the past. Finally, our data show that human lateralization patterns do not align with trends found among other anthropoids, suggesting that unique selective pressures gave rise to the unusual hand preferences displayed by our species.


title: Substrate Specificity for Human Histidine Methyltransferase SETD3

abstract: Histidine methyltransferase SETD3 plays an important role in human biology and diseases. Previously, we showed that SETD3 catalyzes N3-methylation of histidine 73 in {beta}-actin (Kwiatkowski et al., 2018). Here we report integrated synthetic, biocatalytic, biostructural and computational analyses on human SETD3-catalyzed methylation of {beta}-actin peptides possessing histidine and its structurally and chemically diverse mimics. Our enzyme assays supported by biostructural analyses demonstrate that SETD3 has a broader substrate scope beyond histidine, including N-nucleophiles on the aromatic and aliphatic side chains. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics and free-energy simulations provide insight into binding geometries and the free energy barrier for the enzymatic methyl transfer to histidine mimics, further supporting experimental data that histidine is the superior SETD3 substrate over its analogs. This work demonstrates that human SETD3 has a potential to catalyze efficient methylation of several histidine mimics, overall providing mechanistic, biocatalytic and functional insight into {beta}-actin histidine methylation by SETD3.

category: biochemistry


title: Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

abstract: The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of  unnatural natural products.


title: The effects of protein crowders on small molecule drug diffusion

abstract:
Crowded environments affect the pharmacokinetics of drug molecules. Here, we investigate how three macromolecular protein crowders, bovine serum albumin, hen egg-white lysozyme and myoglobin, influence the translational diffusion rates and interactions of four low molecular-weight drugs, fluorescein, doxorubicin, glycogen synthase kinase-3 inhibitor SB216763 and quinacrine. Using Fluorescence Recovery After Photo-bleaching in Line mode (Line FRAP), Brownian dynamics simulations and molecular docking, we find that the diffusive behavior of the small molecules is highly affected by self-aggregation, interactions with the proteins, and surface adhesion. Fluorescein diffusion is decreased by protein crowders due to their interactions. On the other hand, for doxorubicin, the presence of protein crowders increases diffusion by reducing surface interactions. SB216763 shows a third scenario, where BSA, but not myoglobin or lysozyme, reduces self-aggregation, resulting in faster diffusion. Quinacrine was the only small molecule whose diffusion was not affected by the presence of protein crowders. The mechanistic insights gained here into the effects of interactions of small molecules with proteins and surfaces on the translational diffusion of small molecules can assist in optimizing the design of compounds for higher mobility and lower occlusion in complex macromolecular environments.

Significance statementThe activity of small molecules is directly related to their active concentration. This, in turn, relates to their molar concentration and their activity coefficient. Any deviations from ideal behavior affects the small molecule activity. This is particularly important for drugs, where we seek to optimize their molar activity. Here, we follow the diffusional activity of four small molecule drugs in various solutions, with and without protein crowders. The study, which involves experimental measurements and theoretical simulations, shows that 3 of the 4 drugs do not diffuse normally, either due to aggregation, protein binding or surface adhesion. We show the mechanisms driving the abnormal diffusion, and how it can be reduced. Lessons learned from this study can be implemented into drug design.


title: TXNRD3 supports male fertility via the redox control of spermatogenesis

abstract: Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.


title: Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

abstract:
Several hundred billion to a trillion cells die in the body every day and release cell free chromatin particles (cfChPs) which enter into the circulation, or are released locally into extracellular compartments of the body. We have reported that cfChPs from the dying cells can readily enter into living cells and damage their DNA. To test the hypothesis that internalised cfChPs might also inflict mitochondrial damage, we treated NIH3T3 mouse fibroblast cells with cfChPs isolated from sera of healthy individuals (10ng), or co-cultured the cells with hypoxia induced dying NIH3T3 cells. Abundant cfChPs could be detected in the cytoplasm of the treated cells by 4h. The latter was associated with evidence of mitochondrial damage in the form of ultra-structural changes, increased mitochondrial mass, alterations in mitochondrial shape, upregulation of the mitochondrial outer membrane protein TOM20, and changes in mitochondrial membrane potential. We also detected increased fluorescence signals of {gamma}-H2AX and p-ATM signifying double-strand breaks in mitochondrial DNA. There was marked increase in production of mitochondrial superoxide (ROS) as detected by MitoSOX Red, and activation of the intracellular antioxidant enzyme superoxide dismutase-1. Mitochondrial damage and ROS production could be inhibited by a cfChPs deactivating agent viz. anti-histone antibody complexed nanoparticles. Given that 1x109-1x1012 cells die in the body every day, we propose that cfChPs are major physiological triggers for mitochondrial damage and ROS production with an important bearing on human health and disease. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions that have been linked to oxidative stress.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/474529v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1111c19org.highwire.dtl.DTLVardef@4bd7eaorg.highwire.dtl.DTLVardef@14c47e7org.highwire.dtl.DTLVardef@104a4e6_HPS_FORMAT_FIGEXP  M_FIG C_FIG Legend: Cell-free chromatin particles (cfChPs) that circulate in blood, and those that are released locally from dying cells, can readily enter into healthy cells to damage their mitochondria leading to ROS production.

HighlightsO_LICell-free chromatin particles (cfChPs) are continuously released from dying cells
C_LIO_LIcfChPs can readily enter into living cells
C_LIO_LIThe internalised cfChPs inflict physical damage to mitochondria
C_LIO_LIcfChPs induced mitochondrial damage results in ROS production and oxidative stress
C_LIO_LIcfChPs are novel triggers for ROS production with implications for health and disease
C_LI


title: Bioprinting the Tumor Microenvironment with an Upgraded Consumer Stereolithographic 3D Printer

abstract:
A widespread application of three-dimensional (3D) bioprinting in basic and translational research requires the accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithographic (SL) printer was developed. This SL bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a highly parallelized tissue production.

To showcase the capability of the bioprinter, we produced constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel. A thorough characterization of different GelMA/PEGDA ratios revealed that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirmed that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.

category: bioengineering


title: Efficient and Explainable Deep Neural Networks for Airway Symptom Detection in Support of Wearable Health Technology

abstract: Mobile health wearables are often embedded with small processors for signal acquisition and analysis. These embedded wearable systems are, however, limited with low available memory and computational power. Advances in machine learning, especially deep neural networks (DNNs), have been adopted for efficient and intelligent applications to overcome constrained computational environments. In this study, evolutionary optimized DNNs were analyzed to classify three common airway-related symptoms, namely coughs, throat clears and dry swallows. As opposed to typical microphone-acoustic signals, mechanoacoustic data signals, which did not contain identifiable speech information for better privacy protection, were acquired from laboratory-generated and publicly available datasets. The optimized DNNs had a low footprint of less than 150 kB and predicted airway symptoms of interests with 83.7% accuracy on unseen data. By performing explainable AI techniques, namely occlusion experiments and class activation maps, mel-frequency bands up to 8,000 Hz were found as the most important feature for the classification. We further found that DNN decisions were consistently relying on these specific features, fostering trust and transparency of proposed DNNs. Our proposed efficient and explainable DNN is expected to support edge computing on mechano-acoustic sensing wearables for remote, longterm monitoring of airway symptoms.


title: DENA: training an authentic neural network model using Nanopore sequencing data of Arabidopsis transcripts for detection and quantification of N6-methyladenosine on RNA

abstract: Models developed using Nanopore direct RNA sequencing data from in vitro synthetic RNA with all adenosine replaced by N6-methyladenosine (m6A), are likely distorted due to superimposed signals from saturated m6A residues. Here, we develop a neural network, DENA, for m6A quantification using the sequencing data of in vivo transcripts from Arabidopsis. DENA identifies 90% of miCLIP-detected m6A sites in Arabidopsis, and obtains modification rates in human consistent to those found by SCARLET, demonstrating its robustness across species. We sequence the transcriptome of two additional m6A-deficient Arabidopsis, mtb and fip37-4, using Nanopore and evaluate their single-nucleotide m6A profiles using DENA.

category: bioinformatics


title: Protein interaction analysis of Plasmodium falciparum circumsporozoite protein variants with human immunoproteins explains RTS,S vaccine efficacy in Ghana

abstract:
BackgroundThe worlds first malaria vaccine RTS,S provides only partial protection against Plasmodium falciparum infections. The explanation for such low efficacy is unclear. This study examined the associations of parasite genetic variations with binding affinity to human immunological proteins including human leukocyte antigen (HLA) and T cell receptors (TCR) involved in RTS,S-induced immune responses.

MethodsMultiplicity of infections was determined by amplicon deep sequencing of merozoite surface protein 1 (PfMSP1). Genetic variations in the C-terminal of circumsporozoite protein (PfMSP1) gene were examined across 88 samples of P. falciparum collected from high and low transmission settings of Ghana. Binding interactions of PfMSP1 variants and HLA/TCR were analyzed using NetChop and HADDOCK predictions. Anti-CSP IgG levels were measured by ELISA in a subset of 10 samples.

FindingsHigh polyclonality was detected among P. falciparum infections. A total 27 CSP haplotypes were detected among samples. A significant correlation was detected between the CSP and MSP multiplicity of infection (MOI). No clear clustering of haplotypes was observed by geographic regions. The number of genetic differences in PfCSP between 3D7 and non-3D7 variants does not influence binding interactions to HLA/T cells nor anti-CSP IgG levels. Nevertheless, PfCSP peptide length significantly affects its molecular weight and binding affinity to the HLA.

InterpretationsThe presence of multiple non-3D7 strains among P. falciparum infections in Ghana impact the effectiveness of RTS,S. Longer PfCSP peptides may elicit a stronger immune response and should be considered in future version RTS,S. The molecular mechanisms of RTS,S cell-mediated immune responses related to longer CSP peptides warrants further investigations.


title: Equivalence of the Transition Heat Capacities of Proteins and DNA

abstract: It has been reported for many globular proteins that the native heat capacity at 25{degrees}C, per gram, is the same. This has been interpreted to indicate that heat capacity is a fundamental property of native proteins that provides important information on molecular structure and stability. Heat capacities for both proteins and DNA has been suggested to be related to universal effects of hydration/solvation on native structures. Here we report on results from thermal denaturation analysis of two well-known proteins, human serum albumin and lysozyme, and a short DNA hairpin. The transition heat capacities at the Tm for the three molecules were quantitatively evaluated by differential scanning calorimetry. When normalized per gram rather than per mol the transition heat capacities were found to be precisely equivalent. This observation for the transition heat capacities of the proteins is consistent with previous reports. However, an identical transition heat capacity for DNA has not been reported and was unexpected. Further analysis of the collected data suggested a mass dependence of hydration effects on thermal denaturation that is preserved at the individual protein amino acid and DNA base levels. Equivalence of transition heat capacities suggests the possibility of a universal role of hydration effects on the thermal stability of both proteins and DNA.

category: biophysics


title: Structural insights into the ion selectivity of the MgtE channel for Mg2+ over Ca2+

abstract: MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 [A] resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ [~]500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 [A] resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.


title: Extreme value analysis of intracellular cargo transport by motor proteins

abstract: Extreme-value analysis (EVA) deals with deviations of data from the median of probability distributions. It has been used for various purposes, such as predicting disasters and analyzing sports records. Herein, we extended the use of EVA to investigate nanoscale runners within cells. Motor proteins such as kinesin and dynein run along microtubules, which are protein-containing rails, to deliver cargo (material needed for cells). While the velocity of these runners is markedly affected by large cargos in viscous intracellular environments, the return-level EVA plots showed that the velocity near no-load conditions disclosed the physical difference between the two runners, resulting from their load (force)-velocity relationships. The concave-up force-velocity relationship of dynein hampered the independent and identically distributed conditions for the retrograde velocity dataset and the abnormality of the return-level plot of EVA. Our findings underscore the importance of EVA for assessing the physical properties of proteins in vivo.


title: Sequestration of histidine kinases by non-cognate response regulators establishes a threshold level of stimulation for bacterial two-component signaling

abstract: Two-component signaling systems (TCSs) in bacteria are often positively auto-regulated, where the histidine kinase (HK) and response regulator (RR) proteins comprising a TCS are expressed downstream of the signal they transduce. This auto-regulation improves the sensitivity of the TCS to stimuli and amplifies adaptive responses. The downside, however, is that the TCS may mount disproportionately large responses to weak or fleeting signals. How bacteria prevent such disproportionate responses is not known. Here, we show that sequestration of phosphorylated HKs by non-cognate RRs serves as a design to prevent such disproportionate responses. Using TCSs of M. tuberculosis as model systems, we found that with every one of the five HKs we studied, there was at least one non-cognate RR with higher affinity than that of the cognate RR for the HK. Phosphorylated HKs would thus preferentially bind the non-cognate RRs, suppressing signal transduction through the cognate pathways, which we demonstrated in vitro. Using mathematical modeling of TCS signaling in vivo, we predicted that this sequestration would introduce a threshold level of stimulation for a significant response, preventing responses to signals below this threshold. Finally, we showed in vivo using tunable expression systems in M. bovis that upregulation of a higher affinity non-cognate RR substantially suppressed the output from the cognate TCS pathway, presenting strong evidence of sequestration by non-cognate RRs as a design to regulate TCS signaling. Blocking this sequestration may be a novel intervention strategy, as it would compromise bacterial fitness by letting it respond unnecessarily to signals.

category: systems biology


title: Map-independent representation of an aggression-promoting social cue in the main olfactory pathway

abstract: AO_SCPLOWBSTRACTC_SCPLOWWhile the olfactory system is required for proper social behaviors, the molecular basis for how social cues are detected via the main olfactory pathway of mammals is not well-characterized. Trimethylamine is a volatile, sex-specific odor found in adult male mouse urine that selectively activates main olfactory sensory neurons that express trace amine-associated receptor 5 (TAAR5). Here we show that trimethylamine, acting via TAAR5, elicits state-dependent attraction or aversion in male mice and drives inter-male aggression. Genetic knockout of TAAR5 significantly reduces aggression-related behaviors, while adding trimethylamine augments aggressive behavior towards juvenile males. We further show that transgenic expression of TAAR5 specifically in olfactory sensory neurons rescues aggressive behaviors in knockout mice, despite extensive remapping of TAAR5 projections to the olfactory bulb. Our results identify a specific main olfactory input that detects a prominent male-specific odor to induce inter-male aggression in a mammalian species and reveal that apparently innate behavioral responses are independent of patterned glomerular input to the olfactory bulb.

category: neuroscience


title: Properties and proximity proteomics of synaptopodin provide insight into the molecular organization of the spine apparatus of dendritic spines

abstract:
The spine apparatus is a specialization of the neuronal ER in dendritic spines consisting of stacks of interconnected cisterns separated by a dense matrix. Synaptopodin, a specific actin binding protein of the spine apparatus, is essential for its formation, but the underlying mechanisms remain unknown. We show that synaptopodin, when expressed in fibroblasts, forms actin-rich structures with connections to the ER, and that an ER-tethered synaptopodin assembles into liquid condensates. We also identified protein neighbors of synaptopodin in spines by in vivo proximity biotinylation. We validated a small subset of such proteins and showed that they co-assemble with synaptopodin in living cells. One of them is Pdlim7, an actin binding protein not previously identified in spines, and we show its precise colocalization with synaptopodin. We suggest that the matrix of the spine apparatus has the property of a liquid protein condensate generated by a multiplicity of low affinity interactions.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/474557v1_ufig1.gif" ALT="Figure 1">
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title: Information encoded in volumes and areas of dendritic spines is nearly maximal across mammalian brains

abstract:
Long-term information associated with neuronal memory resides in dendritic spines. However, spines can have a limited size due to metabolic and neuroanatomical constraints, which should effectively limit the amount of encoded information in excitatory synapses. This study investigates how much information can be stored in the sizes of dendritic spines, and whether is it optimal in any sense? It is shown here, using empirical data for several mammalian brains across different regions and physiological conditions, that dendritic spines nearly maximize entropy contained in their volumes and surface areas for a given mean size. This result is essentially independent of the type of a fitting distribution to size data, as both short- and heavy-tailed distributions yield similar nearly 100% information efficiency in the majority of cases, although heavy-tailed distributions slightly better fit the data. On average, the highest information is contained in spine volume, and the lowest in spine length or spine head diameter. Depending on a species and brain region, a typical spine can encode between 6.1 and 10.8 bits of information in its volume, and 3.1-8.1 bits in its surface area. Our results suggest a universality of entropy maximization in spine volumes and areas, which can be a new principle of memory storing in synapses.

Significance statementIt is believed that memory in the brain is stored in the parts of excitatory synapses called dendritic spines. But how efficient is the memory capacity given synaptic size variability? Generally, the larger the synapse the more information can be packed in its structure. However, this process comes at some cost, as larger synapses use more metabolic energy and brain tissue. Thus, from a theoretical point of view, there exists a benefit-cost trade-off for storing long-term information in dendritic spines, with a unique optimal solution set by an average spine size. We show that volumes and areas of actual spines in different parts of the brain across different mammals are very close to that optimal theoretical solution for storing information. This means that synaptic information is maximally optimized regardless of brain size, region, and physiological condition, which is a remarkable result.


title: Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation

abstract: Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialised parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae, fungi) like some parasites, but then devour their cell contents by phagocytosis. Besides prey recognition and attachment, this complex behaviour involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure. Yet, the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder, and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g. locomotion, cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g. lytic polysaccharide monooxygenases) belong to families that are largely uncharacterised, emphasising the potential of non-fungal micro-eukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behaviour, and new directions for cell biology research on non-model eukaryotes.

category: microbiology


title: pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

abstract: Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopes dipolespread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Greens tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14{degrees} orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

category: molecular biology


title: Macrophages break interneuromast cell quiescence by intervening the inhibition of Schwann cells in zebrafish lateral line

abstract: In the zebrafish lateral line system, interneuromast cells (INCs) between neuromasts are normally kept quiescent by underlying Schwann cells (SWCs). Upon severe injuries that cause the complete loss of an entire neuromast, INCs can occasionally differentiate into neuromasts but how they escape from the inhibition by SWCs is still unclear. Using a genetic/chemical method to specifically ablate a neuromast, we found a small portion of larvae can regenerate a new neuromast, but the regeneration was hindered by inhibiting macrophages. By in toto imaging, we further discovered heterogeneities in macrophage behavior and distribution along lateral line. We witnessed the crawling of macrophages in between injured lateral line and SWCs during regeneration and also in between the second primordium and the first mature lateral line during development. It implies that macrophages may physically separate and alleviate the inhibition from pLLn and SWCs to break the quiescence of INCs during regeneration and development in the zebrafish lateral line.

category: developmental biology


title: Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of  Corynebacterium glutamicum  at oscillating pH values

abstract: In large-scale bioreactors, gradients in cultivation parameters such as oxygen, substrate and pH result in fluctuating environments. pH fluctuations were identified as a critical parameter for bioprocess performance. Traditionally, scale-down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strain and thus process performance. Here, we demonstrate the application of dynamic microfluidic single-cell cultivation (dMSCC) as a novel scale-down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model parameter. A detailed comparison between two-compartment reactor (two-CR) scale-down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 ([~] 8 min) and disturbed pH 6 ([~]2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two-CR 27%). Afterward, systematic experiments at different symmetric and asymmetric pH oscillations between pH ranges of 4-6 and 8-11 and different intervals from 1 minute to 20 minutes, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions within large-scale bioprocesses, which is difficult to conduct using two-CRs.


title: A nanopore interface for high bandwidth DNA computing

abstract: DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this methods utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.


title: Tissue Registration and Exploration User Interfaces in support of a Human Reference Atlas

abstract: Several international consortia are collaborating to construct a human reference atlas, which is a comprehensive, high-resolution, three-dimensional atlas of all the cells in the healthy human body. Laboratories around the world are collecting tissue specimens from donors varying in sex, age, ethnicity, and body mass index. However, integrating and harmonizing tissue data across 20+ organs and more than 15 bulk and spatial single-cell assay types poses diverse challenges. Here we present the software tools and user interfaces developed to annotate ("register") and explore the collected tissue data. A key part of these tools is a common coordinate framework, which provides standard terminologies and data structures for describing specimens, biological structures, and spatial positions linked to existing ontologies. As of December 2021, the "registration" user interface has been used to harmonize and make publicly available data on 6,178 tissue sections from 2,698 tissue blocks collected by the Human Biomolecular Atlas Program, the Stimulating Peripheral Activity to Relieve Conditions program, the Human Cell Atlas, the Kidney Precision Medicine Project, and the Genotype Tissue Expression project. The second "exploration" user interface enables consortia to evaluate data quality and coverage, explore tissue data in the context of the human body, and guide data acquisition.


title: Impact of circadian time of dosing on cardiac-autonomous effects of glucocorticoids

abstract: Mitochondrial capacity is critical to adapt the high energy demand of the heart to circadian oscillations and diseased states. Glucocorticoids regulate the circadian cycle of energy metabolism, but little is known about how circadian timing of exogenous glucocorticoid dosing directly regulates heart metabolism through cardiomyocyte-autonomous mechanisms. While chronic oncedaily intake of glucocorticoids promotes metabolic stress and heart failure, we recently discovered that intermittent once-weekly dosing of exogenous glucocorticoids promoted muscle metabolism in normal and obese skeletal muscle. However, the effects of glucocorticoid intermittence on heart metabolism and heart failure remain unknown. Here we investigated the extent to which circadian time of dosing regulates the effects of the glucocorticoid prednisone in heart metabolism and function in conditions of single pulse or chronic intermittent dosing. In WT mice, we found that prednisone improved cardiac content of NAD+ and ATP with light-phase dosing (ZT0), while the effects were blocked by dark-phase dosing (ZT12). The drug effects on mitochondrial function were cardiomyocyte-autonomous, as shown by inducible cardiomyocyte-restricted glucocorticoid receptor (GR) ablation, and depended on an intact cardiomyocyte clock, as shown by inducible cardiomyocyte-restricted ablation of Brain and Muscle ARNT-like 1 (BMAL1). Conjugating time-of-dosing with chronic intermittence, we found that once-weekly prednisone improved metabolism and function in heart after myocardial injury dependent on circadian time of intake, i.e. with lightphase but not dark-phase dosing. Our study identifies cardiac-autonomous mechanisms through which circadian-specific intermittent dosing reconverts glucocorticoid drugs to metabolic boosters for the heart.

category: physiology


title: Impact of circadian time of dosing on cardiomyocyte-autonomous effects of glucocorticoids


title: High density single-unit human cortical recordings using the Neuropixels probe

abstract:
A fundamental unit of neural computation is the action potential. While significant advances have been made in the ability to sample action potentials of large numbers of individual neurons in animal models, translation of these methodologies to humans has been lacking due to clinical time constraints, electrical noise in the operating room, and reliability of the methodology. Here we present a reliable method for intraoperative recording of dozens of neurons in humans using the Neuropixels probe, yielding up to [~]100 simultaneously-recorded single-units (n=596 across 11 recordings in 8 participants). Most single-units were active within 1 minute of reaching target depth, compatible with clinical time constraints. Cell pairs active close in time were spatially closer in most recordings, demonstrating the power to resolve complex cortical dynamics. Altogether, this approach provides access to population single-unit activity across the depth of human neocortex at scales previously only accessible in animal models.

HighlightsO_LISingle units in 8 patients, yielding 596 putative single units across 11 recordings
C_LIO_LIThe majority of putative neurons fire at least 1 spike within one minute after reaching target depth
C_LIO_LIPutative neurons take longer to fire at least one spike in anesthetized compared to awake participants
C_LIO_LICell-pairs that fire action potentials close in time are spatially closer together than those fire further apart in time
C_LI


title: Work and oxygen consumption of isolated right ventricular papillary muscle in experimental pulmonary hypertension

abstract:
Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis was tested that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular (RV) muscle preparations were subjected to 5 Hz sinusoidal length changes at 37{degrees}C. Work and suprabasal oxygen consumption (VO2) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane (PP IMM), and monoamine oxidase (MAO)-A and glucose 6-phosphate dehydrogenase (G-6-PDH) activities and phosphatidylglycerol (PG) and cardiolipin (CL) contents were determined. Mechanical efficiency ranged from 23 to 11% in control (n = 6) and from 22 to 1% in MCT-PH (n = 15) and correlated with work (r2 = 0.68, P < 0.0001) but not with VO2 (r2 = 0.004, P = 0.7919). VO2 for cross-bridge cycling was proportional to work (r2 = 0.56, P = 0.0005). Blebbistatin-resistant VO2 (r2 = 0.32, P = 0.0167) and IMM PP (r2 = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination R2 = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2 = 0.28, P = 0.0391), but not with efficiency (r2 = 0.20, P = 0.0867). G-6-PDH, MAO-A and PG/CL increased in the RV wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is due to activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant VO2 are discussed.

Key pointsO_LIMechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown.
C_LIO_LIWork and oxygen consumption is determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements.
C_LIO_LICytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension.
C_LIO_LIMitochondrial dysfunction in right ventricular myocytes is related to reduced work and mechanical efficiency in experimental pulmonary hypertension.
C_LIO_LIUpregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is due to excessive production of reactive oxygen species.
C_LI


title: Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement

abstract: The SARS-CoV-2 Omicron variant of concern evades antibody mediated immunity with an unprecedented magnitude due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo-electron microscopy and X-ray crystal structures of the spike and RBD bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a structural framework for understanding the marked reduction of binding of all other therapeutic mAbs leading to dampened neutralizing activity. We reveal electrostatic remodeling of the interactions within the spike and those formed between the Omicron RBD and human ACE2, likely explaining enhanced affinity for the host receptor relative to the prototypic virus.


title: Endosomal structure and APP biology are not altered in preclinical cellular models of Down syndrome

abstract: Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimers disease - dementia. Alzheimers disease is characterised by the accumulation in the brain of amyloid-{beta} plaques that are a product of amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-{beta} accumulation is within endosomes and changes to endosome biology occur early in disease. Here we determine if primary mouse embryonic fibroblasts isolated from two mouse models of Down syndrome can be used to study endosome and APP cell biology. We report that in these cellular models of Down syndrome endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in these models, despite three copies of App.


title: Differential regulation of prelimbic and thalamic transmission to the basolateral amygdala by acetylcholine receptors

abstract:
The amygdalar anterior basolateral nucleus (BLa) plays a vital role in emotional behaviors. This region receives dense cholinergic projections from basal forebrain which are critical in regulating neuronal activity and synaptic transmission. Cholinergic signaling in BLa is thought to occur through both a slow mode of volume transmission as well as a rapid, phasic mode. However, the relative effect of each mode of signaling in BLa is not understood. Here, we used electrophysiology and optogenetics in mouse brain slices to compare regulation of afferent input from cortex and thalamus to the BLa by these two modes of transmission. Phasic ACh release evoked by single pulse stimulation of cholinergic terminals had a biphasic effect on glutamatergic transmission at cortical input, producing rapid nicotinic receptor-mediated facilitation followed by slower muscarinic receptor (mAChR)-mediated depression. In contrast, tonic elevation of ACh through application of the cholinesterase inhibitor physostigmine suppressed glutamatergic transmission at cortical inputs through mAChRs only. This suppression was not observed at thalamic inputs to BLa. In agreement with this pathway-specificity, the mAChR agonist, muscarine more potently suppressed transmission at inputs from prelimbic cortex (PL) than thalamus. Muscarinic inhibition at PL input was dependent on presynaptic M4 mAChRs, while at thalamic input it depended upon M3 mAChR-mediated stimulation of retrograde endocannabinoid signaling. Muscarinic inhibition at both pathways was frequency-dependent, allowing only high frequency activity to pass. These findings demonstrate complex cholinergic regulation of afferent input to BLa that depends upon the mode of ACh release and is both pathway specific and frequency dependent.

Significance statementCholinergic modulation of the basolateral amygdala regulates formation of emotional memories, but the mechanisms underlying this regulation are not well understood. Here, we show, using mouse brain slices, that ACh differentially regulates afferent transmission to the BLa depending on the mode of cholinergic signaling. Rapid, phasic ACh produces a biphasic excitatory-inhibitory regulation of glutamatergic transmission mediated by nicotinic and muscarinic receptors, respectively. In contrast, slow, tonic ACh produces muscarinic inhibition only. Tonic regulation is pathway specific with cortical input regulated more strongly than thalamic input. This disparity is caused by differential regulation by M4 and M3 receptors at the two inputs. Specific targeting of these receptors may thus provide a therapeutic strategy to bias amygdalar processing and regulate emotional memory.


title: Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells

abstract: Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to non-peptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

category: immunology


title: A Xenopus neuromast bioassay for chemical ototoxicity

abstract:
BackgroundOtotoxic chemicals can impair the senses of hearing and balance in mammals through irreversible damage to the mechanosensory bundles of inner ear hair cells. Fish and amphibians are useful models for investigating ototoxicity because their inner ear hair cells, like those of mammals, are susceptible to damage by ototoxins. Moreover, amphibian mechanosensation is augmented by a lateral line organ on the body surface that comprises external mechanosensory hair cells. The lateral line hair cells are arranged in clusters (neuromasts) and are structurally and functionally similar to inner ear hair cells, but are more accessible for experimental manipulation. Herein, we implemented neuromasts of the amphibian (Xenopus) lateral line as an organ system for evaluating the effects of ototoxic chemicals, such as antibiotics, on mechanosensory hair cell bundles.

MethodsWe examined the ultrastructure of larval Xenopus laevis neuromasts with scanning electron microscopy (SEM) after larvae were continuously exposed to ototoxic aminoglycoside antibiotics at sub-lethal concentrations (gentamicin; streptomycin; neomycin) for 72 hours.

ResultsSEM images demonstrated that 72 hours of exposure to antibiotic concentrations greater than 25 {micro}M reduced the hair cell bundle number in lateral line neuromasts.

ConclusionTherapeutic drug studies will benefit from the incorporation of bioassay strategies that evaluate ototoxicity across multiple species including genera of amphibian origin such as Xenopus. Our outcomes support the use of the Xenopus lateral line for identification of potential ototoxic chemicals and suggest that Xenopus neuromast hair cell bundles can withstand antibiotic exposure. The Xenopus bioassay presented here can be incorporated into drug discovery methodology as a high-resolution phenotypic screen for ototoxic effects.

Summary statementDamage to sensory cells of the inner ear by chemical agents such as antibiotics contributes to the growing global prevalence of disorders of hearing and balance. Our results demonstrate that the Xenopus lateral line, in conjunction with SEM, affords an accessible organ system for otoxicity screens during the drug discovery pipeline.


title: Roles for Mitochondrial Complex I subunits in regulating synaptic transmission and growth

abstract: To identify conserved components of synapse function that are also associated with human diseases, we conducted a genetic screen. We used the Drosophila melanogaster neuromuscular junction (NMJ) as a model. We employed RNA interference (RNAi) on selected targets and assayed synapse function and plasticity by electrophysiology. We focused our screen on genetic factors known to be conserved from human neurological or muscle functions (300 Drosophila lines screened). From our screen, knockdown of a Mitochondrial Complex I (MCI) subunit gene (ND-20L) lowered levels of NMJ neurotransmission. Due to the severity of the phenotype, we studied MCI function further. Knockdown of core MCI subunits concurrently in neurons and muscle led to impaired neurotransmission. We localized this neurotransmission function to the muscle. Pharmacology targeting MCI phenocopied the impaired neurotransmission phenotype. Finally, MCI subunit knockdowns or pharmacological inhibition led to profound cytological defects, including reduced NMJ growth and altered NMJ morphology. Mitochondria are essential for cellular bioenergetics and produce ATP through oxidative phosphorylation. Five multi-protein complexes achieve this task, and MCI is the largest. Impaired Mitochondrial Complex I subunits in humans are associated with disorders such as Parkinsons disease, Leigh syndrome, and cardiomyopathy. Together, our data present an analysis of Complex I in the context of synapse function and plasticity. We speculate that in the context of human MCI dysfunction, similar neuronal and synaptic defects could contribute to pathogenesis.


title: In silico Elucidation of Dihydroquinine Mechanism of Action against Toxoplasma gondii

abstract: Dihydroquinine (DHQ), is a quinine-based compound with anti-malarial properties. However, little is known about its mechanism of action against T. gondii inhibition, which shares similar biology with Plasmodium spp. In order to explore DHQ activity as an inhibitor of T. gondii using in vitro assays, we first used an in silico approach to decipher its mechanisms of action based on previous knowledge about its disruption of nucleic acid and protein synthesis. An in silico study was performed on T. gondii parasite replication, transcriptional and translational machinery to decipher the binding potentials of DHQ to some top selected enzymes. We report for the first time, using an in silico analysis that showed that DHQ binds strongly to DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase and thus could affect DNA replication, transcriptional and translational activities in T. gondii. Also, we found DHQ to effectively bind to mitochondria detoxifying enzymes (i.e., superoxide dismutase (SOD), peroxidoxin, and Catalase (CAT)). In conclusion, DHQ could be a lead compound for the treatment of toxoplasmosis when successfully evaluated using in vitro and in vivo models to confirm its effectiveness and safety.

category: cell biology


title: The gut microbiota affects the social network of honeybees

abstract: The gut microbiota influences animal neurophysiology and behavior but has not previously been documented to affect emergent group-level behaviors. Here we combine gut microbiota manipulation with automated behavioral tracking of honeybee sub-colonies to show that the microbiota increases the rate and specialization of social interactions. Microbiota colonization was associated with higher abundances of one third of metabolites detected in the brain, including several amino acids, and a subset of these metabolites were significant predictors of social interactions. Colonization also affected brain transcriptional processes related to amino acid metabolism and epigenetic modification in a brain region involved in sensory perception. These results demonstrate that the gut microbiota modulates the emergent colony social network of honeybees, likely via changes in chromatin accessibility and amino acid biosynthesis.

category: animal behavior and cognition


title: Gut Microbial Trimethylamine is Elevated in Alcohol-Associated Hepatitis and Contributes to Ethanol-Induced Liver Injury in Mice

abstract:
BACKGROUNDThere is mounting evidence that microbes resident in the human intestine contribute to diverse alcohol-associated liver diseases (ALD) including the most deadly form known as alcohol-associated hepatitis (AH). However, mechanisms by which gut microbes synergize with excessive alcohol intake to promote liver injury are poorly understood. Furthermore, whether drugs that selectively target gut microbial metabolism can improve ALD has never been tested.

METHODSWe used liquid chromatography tandem mass spectrometry to quantify the levels of microbe and host choline co-metabolites in healthy controls and AH patients, finding elevated levels of the microbial metabolite trimethylamine (TMA) in AH. In subsequent studies, we treated mice with non-lethal bacterial choline TMA lyase (CutC/D) inhibitors to blunt gut microbe-dependent production of TMA in the context of chronic ethanol administration. Indices of liver injury were quantified by complementary RNA sequencing, biochemical, and histological approaches. In addition, we examined the impact of ethanol consumption and TMA lyase inhibition on gut microbiome structure via 16S rRNA sequencing.

RESULTSWe show the gut microbial choline metabolite trimethylamine (TMA) is elevated in AH patients and correlates with reduced hepatic expression of the TMA oxygenase flavin-containing monooxygenase 3 (FMO3). Provocatively, we find that small molecule inhibition of gut microbial CutC/D activity protects mice from ethanol-induced liver injury. CutC/D inhibitor-driven improvement in ethanol-induced liver injury is associated with distinct reorganization of the gut microbiome and host liver transcriptome.

CONCLUSIONSThe microbial metabolite TMA is elevated in patients with AH, and inhibition of TMA production from gut microbes can protect mice from ethanol-induced liver injury.


title: DNA origami as a nanomedicine for targeted rheumatoid arthritis therapy through reactive oxygen species and nitric oxide scavenging

abstract: High levels of reactive oxygen species (ROS) and nitric oxide (NO) generated by M1 macrophages induce inflammation in the development of rheumatoid arthritis (RA). The eliminating of ROS and NO therefore represents an alternative strategy for RA treatment. Because DNA molecules possess ROS- and endogenous NO-scavenging capability, herein, we develop a nanomedicine based on triangular DNA origami nanostructures for targeted RA treatment. We showed that folic acid-modified triangular DNA origami nanostructures (FA-tDONs) could reduce ROS and NO simultaneously inside proinflammatory M1 macrophages, leading to their polarization into anti-inflammatory M2 subtype. Further in vivo studies confirmed that FA-tDONs could actively target inflamed joints in collagen-induced arthritis (CIA) mice, attenuate inflammatory cytokines and alleviate disease progression. This work demonstrated that DNA origami itself could act as a potential nanomedicine for targeted RA treatment.


title: Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

abstract: The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.


title: Loss of p53 tumor suppression function drives invasion and genomic instability in models of murine pancreatic cancer

abstract: Pancreatic ductal adenocarcinoma (PDA) is a deadly disease with few treatment options. There is an urgent need to better understand the molecular mechanisms that drive disease progression, with the ultimate aim of identifying early detection markers and clinically actionable targets. To investigate the transcriptional and morphological changes associated with pancreatic cancer progression, we analyzed the KrasLSLG12D/+; Trp53LSLR172H/+; Pdx1-Cre (KPC) mouse model. We have identified an intermediate cellular event during pancreatic carcinogenesis in the KPC mouse model of PDA that is represented by a subpopulation of tumor cells that express KrasG12D, p53R172H and one allele of wild-type Trp53. In vivo, these cells represent a histological spectrum of pancreatic intraepithelial neoplasia (PanIN) and acinar-to-ductal metaplasia (ADM) and rarely proliferate. Following loss of wild-type p53, these precursor lesions undergo malignant de-differentiation and acquire invasive features. We have established matched organoid cultures of pre-invasive and invasive cells from murine PDA. Expression profiling of the organoids led to the identification of markers of the pre-invasive cancer cells in vivo and mechanisms of disease aggressiveness.

category: cancer biology


title: Global ecomorphological restructuring of dominant marine reptiles prior to the K/Pg mass extinction

abstract: Mosasaurid squamates were the dominant amniote predators in marine ecosystems during most of the Late Cretaceous. Evidence from multiple sites worldwide of a global mosasaurid community restructuring across the Campanian-Maastrichtian transition may have wide-ranging implications for the evolution of diversity of these top oceanic predators. In this study, we use a suite of biomechanical traits and functionally descriptive ratios to investigate how the morphofunctional disparity of mosasaurids evolved through time and space prior to the Cretaceous-Palaeogene (K/Pg) mass extinction. Our results suggest that the worldwide taxonomic turnover in mosasaurid community composition from Campanian to Maastrichtian is reflected by a notable increase in morphofunctional disparity on a global scale, but especially driven the North American record. Ecomorphospace occupation becomes more polarised during the late Maastrichtian, as the morphofunctional disparity of mosasaurids plateaus in the Southern Hemisphere and decreases in the Northern Hemisphere. We show that these changes are not associated with strong modifications in mosasaurid size, but rather with the functional capacities of their skulls, and that mosasaurid morphofunctional disparity was in decline in several provincial communities before the K-Pg mass extinction. Our study highlights region-specific patterns of disparity evolution, and the importance of assessing vertebrate extinctions both globally and regionally. Ecomorphological differentiation in mosasaurid communities, coupled with declines in other formerly abundant marine reptile groups, indicates widespread restructuring of higher trophic levels in marine food webs was well underway when the K-Pg mass extinction took place.

category: paleontology


title: Exercise and the brain: a mechanical model for pulsation on flow of cerebrospinal fluid

abstract: Exchange of molecules between cerebrospinal fluid (CSF) and brain cells contributes to brain function and protection from dementia. Despite widespread acceptance of the  glymphatic theory, the route by which CSF is brought close enough to the neural tissue for solutes to be exchanged by extracellular diffusion is not entirely clear. Exogenous molecules injected into CSF are observed to reach the basement lamina that surrounds the dense capillary network. Transport of solutes by diffusion along the basement lamina, a gel of macromolcules about 100 nm thick, would be too slow; bulk flow in a static geometry would require unphysiologically high pressures. However, it is known that the pulsation of blood aids transport of CSF, and we hypothesized that this is because the pulsation intermittently squeezes the pericapillary lamina. In a primitive mimicry, we have tested whether intermittent squeezing increases flow through an agar gel. In all but one of 216 tests, pulsation caused a reversible increase, sometimes by a factor of 100 or more. The enhancement was greatest for frequencies 5-11 Hz and, over the tested range of pressure heads (20 - 50 cmH2O), was greatest for the lowest pressure. The results suggest a reason why exercise slows the aging of the brain.


title: Intermittent compression increases flow through a gel: a mechanical model perhaps relevant to flow of cerebrospinal fluid


title: Superficial white matter microstructure affects processing speed in cerebral small vessel disease

abstract: White matter hyperintensities (WMH) are a typical feature of cerebral small vessel disease (CSVD). This condition contributes to about 50% of dementias worldwide, a massive health burden in aging. Microstructural alterations in the deep white matter (DWM) have been widely examined in CSVD. However, little is known about abnormalities in the superficial white matter (SWM) and their relevance for processing speed, the main cognitive deficit in CSVD. In this paper, 141 patients with CSVD were studied. Processing speed was assessed by the completion time of the Trail Making Test Part A. White matter abnormalities were assessed by WMH burden (lesion volume on T2-FLAIR) and diffusion MRI, including DTI and free-water (FW) imaging microstructure measures. The results of our study indicate that the superficial white matter may play a particularly important role in cognitive decline in CSVD. SWM imaging measures resulted in a large contribution to processing speed, despite a relatively small WMH burden in the SWM. SWM FW had the strongest association with processing speed among all imaging markers and, unlike the other diffusion MRI measures, significantly increased between two patient subgroups with the lowest WMH burdens (possibly representing early stages of disease). When comparing two patient subgroups with the highest WMH burdens, the involvement of WMH in the SWM was accompanied by significant differences in processing speed and white matter microstructure. Given significant effects of WMH volume and regional FW on processing speed, we performed a mediation analysis. SWM FW was found to fully mediate the association between WMH volume and processing speed, while no mediation effect of DWM FW was observed. Overall, our findings identify SWM abnormalities in CSVD and suggest that the SWM has an important contribution to processing speed. Results indicate that FW in the SWM is a sensitive marker of microstructural changes associated with cognition in CSVD. This study extends the current understanding of CSVD-related dysfunction and suggests that the SWM, as an understudied region, can be a potential target for monitoring pathophysiological processes in future research.


title: A common human brain-derived neurotrophic factor polymorphism leads to sustained depression of excitatory synaptic transmission by isoflurane in hippocampus

abstract: Multiple presynaptic and postsynaptic targets have been identified for the reversible neurophysiological effects of general anesthetics on synaptic transmission and neuronal excitability. However, the synaptic mechanisms involved in persistent depression of synaptic transmission resulting in more prolonged neurological dysfunction following anesthesia are less clear. Here, we show that brain-derived neurotrophic factor (BDNF), a growth factor implicated in synaptic plasticity and dysfunction, enhances glutamate synaptic vesicle exocytosis, and that attenuation of vesicular BDNF release by isoflurane contributes to transient depression of excitatory synaptic transmission in mice. This reduction in synaptic vesicle exocytosis was irreversible in neurons that release less endogenous BDNF due to a polymorphism (BDNF Val66Met) compared to wild-type mouse hippocampal neurons following isoflurane exposure. These effects were prevented by exogenous application of BDNF. Our findings identify a role for a common human BDNF single nucleotide polymorphism (Val66Met; rs6265) in persistent changes of synaptic function following isoflurane exposure. These persistent alterations in excitatory synaptic transmission have important implications for the role of genotype in anesthetic effects on synaptic plasticity and neurocognitive function.


title: Microglia influence neurofilament deposition in ALS iPSC-derived motor neurons

abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motor neuron loss is the primary phenotype, leading to muscle weakness and wasting, respiratory failure, and death. Although a portion of ALS cases are linked to one of over 50 unique genes, the vast majority of cases are sporadic in nature. However, the mechanisms underlying the motor neuron loss in either familial or sporadic ALS are not entirely clear. Here we used induced pluripotent stem cells derived from a set of identical twin brothers discordant for ALS to assess the role of astrocytes and microglia on the expression and accumulation of neurofilament proteins in motor neurons. We found that motor neurons derived from the affected twin exhibited increased transcript levels of all three neurofilament isoforms and increased expression of phosphorylated neurofilament puncta. We further found that treatment of the motor neurons with astrocyte conditioned medium and microglial conditioned medium significantly impacted neurofilament deposition. Together, these data suggest that glial-secreted factors can alter neurofilament pathology in ALS iPSC-derived motor neurons.


title: INFLUENCE OF RAT CENTRAL THALAMIC NEURONS ON FORAGING BEHAVIOR IN A HAZARDOUS ENVIRONMENT

abstract: The basolateral amygdala (BL) is a major regulator of foraging behavior. Following BL inactivation, rats become indifferent to predators. However, at odds with the view that the amygdala detects threats and generate defensive behaviors, most BL neurons have reduced firing rates during foraging and at proximity of the predator. In search of the signals determining this unexpected activity pattern, this study considered the contribution of the central medial thalamic nucleus (CMT), which sends a strong projection to BL, mostly targeting its principal neurons. Inactivation of CMT or BL with muscimol abolished the rats normally cautious behavior in the foraging task. Moreover, unit recordings revealed that CMT neurons showed large but heterogeneous activity changes during the foraging task, with many neurons decreasing or increasing their discharge rates, with a modest bias for the latter. A generalized linear model revealed that CMT neurons encode many of the same task variables as principal BL cells. However, the nature (inhibitory vs. excitatory) and relative magnitude of the activity modulations seen in CMT neurons differed markedly from those of principal BL cells but were very similar to those of fast-spiking BL interneurons. Together, these findings suggest that, during the foraging task, CMT inputs fire some principal BL neurons, recruiting feedback interneurons in BL, resulting in the widespread inhibition of principal BL cells.


title: Muscarinic acetylcholine receptors modulate HCN channel properties in vestibular ganglion neurons

abstract:
Vestibular efferent neurons play an important role in shaping vestibular afferent excitability and, accordingly, on the information encoded by their spike patterns. Efferent- modulation is linked to muscarinic signaling cascades that affect ion channel conductances, most notably low-voltage gated potassium channels such as KCNQ. Here we tested and found that muscarinic signaling cascades also modulate hyperpolarization- activated cyclic-nucleotide gated channels (HCN). HCN channels play a key role in controlling spike-timing regularity and a non-chemical form of transmission between type I hair cells and vestibular afferents. The impact of cholinergic efferent input on HCN channels was assessed using voltage-clamp methods, which measure currents in the disassociated cell bodies of vestibular ganglion neurons (VGN). Membrane properties in VGN were characterized before and after administration of the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M). We found that Oxo-M shifted the voltage-activation range of HCN channels in the positive direction by 3.2 {+/-} 0.7 mV, which more than doubled the available current when held near rest at -70 mV (a 139 {+/-} 43% increase, n=12). This effect was not blocked by pre-treating the cells with a KCNQ channel blocker, linopirdine, which suggests that this effect is not dependent on KCNQ currents. We also found that HCN channel properties in the baseline condition and sensitivity to mAChR activation depended on cell size and firing patterns. Large-bodied neurons with onset firing patterns had the most depolarized activation range and least sensitivity to mAChR activation. Together, our results highlight the complex and dynamic regulation of HCN channels in VGN.

Significance StatementVestibular afferents express a diverse complement of ion channels. This diversity is key to shaping their various response properties, allowing them to encode both fast and slow head movements. In vitro studies that characterized afferent diversity identified low-voltage activated potassium channels and hyperpolarization-activated cyclic- nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Afferent excitability is known to be controlled by a network of acetylcholine-releasing efferent neurons that close a type of low-voltage activated potassium channel found on the afferent neuron. Here, we show that the same efferent signaling cascade that shuts these potassium channels also enhances the activation of HCN channels by depolarizing their voltage-activation range. The size of this effect varies from cell to cell depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system robust and flexible control over the excitability and timing properties vestibular afferent activity.


title: The synaptic organization in the  C. elegans neural network suggests significant local compartmentalized computations

abstract: Neurons are characterized by elaborate tree-like dendritic structures that support local computations by integrating multiple inputs from upstream presynaptic neurons. It is less clear if simple neurons, consisting of a few or even a single neurite, may perform local computations as well. To address this question, we focused on the compact neural network of C. elegans animals for which the full wiring diagram is available, including the coordinates of individual synapses. We find that the positions of the chemical synapses along the neurites are not randomly distributed, nor can they be explained by anatomical constraints. Instead, synapses tend to form clusters, an organization that supports local compartmentalized computations. In mutually-synapsing neurons, connections of opposite polarity cluster separately, suggesting that positive and negative feedback dynamics may be implemented in discrete compartmentalized regions along neurites. In triple-neuron circuits, the non-random synaptic organization may facilitate local functional roles, such as signal integration and coordinated activation of functionally-related downstream neurons. These clustered synaptic topologies emerge as a guiding principle in the network presumably to facilitate distinct parallel functions along a single neurite, effectively increasing the computational capacity of the neural network.


title: Impaired endogenous neurosteroid signaling contributes to behavioral deficits associated with chronic stress

abstract: Chronic stress is a major risk factor for psychiatric illnesses, including depression; however, the pathophysiological mechanisms whereby stress leads to mood disorders remain unclear. The recent FDA approval of antidepressants with novel mechanisms of action, like Zulresso(R), a synthetic neuroactive steroid analog with molecular pharmacology similar to allopregnanolone, has spurred interest in new therapeutic targets and, potentially, novel pathophysiological mechanisms for depression. Allopregnanolone acts as a positive allosteric modulator of GABAA receptors (GABAARS), acting preferentially at {delta} subunit-containing receptors ({delta}-GABAARS). Accumulating clinical and preclinical evidence supports the antidepressant effects of exogenous administration of allopregnanolone and allopregnanolone analogs; however, the role of endogenous neurosteroids in the pathophysiology of depression remains unknown. Here, we examine whether altered neurosteroid signaling may contribute to behavioral deficits following chronic unpredictable stress (CUS) in mice. We first identified reductions in expression of {delta}-GABAARs, the predominant site of action of 5a-reduced neuroactive steroids, following CUS. Additionally, utilizing LC-MS/MS we discovered a decrease in levels of allopregnanolone in the BLA, but not plasma of mice following CUS, an indication of impaired neurosteroid synthesis. CRISPR knockdown the rate-limiting enzymes involved in allopregnanolone synthesis, 5-reductase type 1 and 2, in the BLA mimicked the behavioral deficits associated with CUS in mice. Furthermore, overexpression expression of 5-reductase type 1 and 2 in the BLA improved behavioral outcomes. Collectively, this suggests chronic stress impairs endogenous neurosteroid signaling in the BLA which is sufficient to induce behavioral deficits similar to those observed following CUS. Further, these studies suggest that the therapeutic efficacy of allopregnanolone-based treatments may be due to their ability to directly target the underlying pathophysiology of mood disorders. Therefore, targeting endogenous neurosteroidogenesis may offer a novel therapeutic strategy for the treatment of mood disorders.


title: V1 receptive field structure contributes to neuronal response latency

abstract: The timing of neuronal responses is considered to be important for information transferring and communication across individual neurons. However, the sources of variabilities in the timing of neuronal responses are not well understood and sometimes over-interpreted. A systematic variability in the response latencies of the primary visual cortex has been reported in presence of drifting grating stimulus. Whereas the response latencies are systematically dependent on stimulus orientation. To understand the underlying mechanism of these systematic latencies, we recorded the neuronal response of the cat visual cortex, area 17, and simulated the response latency of V1 neurons, with two geometric models. We showed that outputs of these two models significantly predict the response latencies of the electrophysiology recording during orientation tasks. The periodic patterns created in the raster plots were dependent on the relative position of the stimulus rotation center and the receptive-field sub-regions. We argue the position of stimulus is contributing to systematic response latencies, dependent on drifting orientation. Therefore, we provide a toolbox based on our geometrical model for determining the exact location of RF sub-regions. Our result indicates that a major source of neuronal variability is the lack of fine-tuning in the task parameters. Considering the simplicity of the orientation selectivity task, we argue fine-tuning of stimulus properties is crucial for deduction of neural variability in higher-order cortical areas and understanding their neural dynamics.


title: A systematic characterization of intrinsically formed microglia-like cells during retinal organoid differentiation.

abstract:
Brain organoids differentiated from human induced pluripotent stem cells provide a unique opportunity to investigate the development, organization and connectivity of neurons in a complex cellular environment. However, organoids usually lack microglia, brain-resident immune cells which are both present in the early human embryonic brain and participate in neuronal circuit development.

Here, we find that microglia innately develop in unguided retinal organoid differentiation between week 3 and 4 in 2.5D culture and appear later in floating, non-pigmented, 3D-cystic compartments. We enriched for cystic structures using a low-dosed BMP4 application and performed mass spectrometry, thus defining the protein composition of microglia-containing compartments. We found that cystic compartments expressed both mesenchymal and epithelial markers with microglia enriched in the mesenchymal region. Interestingly, microglia-like cells started to express the border-associated macrophage marker CD163. The preferential localization of human microglia to a mesenchymal compartment provides insight into the behavior and migration of microglia. The model will ultimately allow detailed study of these enigmatic cells and how they enter and distribute within the human brain.


title: Oxytocin modulates social brain network correlations in resting and task state

abstract: Oxytocin (OT) is a neuropeptide that modulates social behaviors and the social brain. The effects of OT on the social brain can be tracked by assessing the neural activity in the resting and task states, providing a system-level framework for characterizing state-based functional relationships of its distinct effect. Here, we contribute to this framework by examining how OT modulates social brain network correlations during the resting and task states using fMRI. Firstly, we investigated network activation, followed by analyzing the relationship between networks and individual differences measured by the Positive and Negative Affect Schedule and the Big-Five scales. Subsequently, we evaluated functional connectivity in both states. Finally, the relationship between networks across the states was represented by the predictive power of networks in the resting state for task-evoked activity. The difference in predicted accuracy between subjects displayed individual variations in this relationship. Our results showed decreased dorsal default mode network (DDMN) for the OT group in the resting state. Additionally, only in the OT group, the activity of the DDMN in the resting state had the largest predictive power for task-evoked activation of the precuneus network (PN). The results also demonstrated OT reduced individual variation of PN, specifically, the difference of accuracy between predicting a subjects own and others PN task activation. These findings together suggest a distributed but modulatory effect of OT on the association between resting brain networks and task-dependent brain networks, showing increased DDMN to PN connectivity after OT administration, which may support OT-induced distributed processing during task performance.


title: Memory persistence and differentiation into antibody-secreting cells accompanied by positive selection in longitudinal BCR repertoires

abstract: The stability and plasticity of B cell-mediated immune memory ensures the ability to respond to the repeated challenges. We have analyzed the longitudinal dynamics of immunoglobulin heavy chain repertoires from memory B cells, plasmablasts, and plasma cells from the peripheral blood of generally healthy volunteers. We reveal a high degree of clonal persistence in individual memory B cell subsets, with inter-individual convergence in memory and antibody-secreting cells (ASCs). ASC clonotypes demonstrate clonal relatedness to memory B cells, and are transient in peripheral blood. We identify two clusters of expanded clonal lineages with differing prevalence of memory B cells, isotypes, and persistence. Phylogenetic analysis revealed signs of reactivation of persisting memory B cell-enriched clonal lineages, accompanied by new rounds of affinity maturation during proliferation and differentiation into ASCs. Negative selection contributes to both persisting and reactivated lineages, preserving the functionality and specificity of BCRs to protect against current and future pathogens.


title: Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

abstract: The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that the complement system reacts with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage susceptibility to fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc, the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during H. capsulatum infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.


title: An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis

abstract: Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA namely, PAX9, MSX1, EDA1, AXIN2 and WNT10A. In this study, an India family with CTA was investigated and a novel c.336C>G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112 amino acid position located at the functionally significant DNA binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. Further RNA-sequencing of PAX9 over-expressed HEK293, identified two potential novel targets, WNT4 and WNT7b those are up-regulated by wild-type PAX9 but not by mutant. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C>G (p.Cys112Trp) variation leads to loss-of-function of PAX9 leading to CTA in this family.


title: New 63 knot and other knots in human proteome from AlphaFold predictions

abstract: AlphaFold is a new, highly accurate machine learning protein structure prediction method that outperforms other methods. Recently this method was used to predict the structure of 98.5% of human proteins. We analyze here the structure of these AlphaFold-predicted human proteins for the presence of knots. We found that the human proteome contains 65 robustly knotted proteins, including the most complex type of a knot yet reported in proteins. That knot type, denoted 63 in mathematical notation, would necessitate a more complex folding path than any knotted proteins characterized to date. In some cases AlphaFold structure predictions are not highly accurate, which either makes their topology hard to verify or results in topological artifacts. Other structures that we found, which are knotted, potentially knotted, and structures with artifacts (knots) we deposited in a database available at: https://knotprot.cent.uw.edu.pl/alphafold.


title: Rsc3K, a SMV resistance gene localized on chromosome 2 in soybean cv. Kefeng No.1, interacts with virulence determinant P3

abstract:
Soybean mosaic virus (SMV) is one of the most devastating viral pathogens in Glycine max (L.) Merr (soybean). Twenty-two SMV strains (SC1-SC22) isolated in China were identified based on their responses to ten soybean cultivars. By using the F2-derived F3 (F2:3) and recombinant inbred line (RIL) populations of resistant Soybean cultivar (cv.) Kefeng No.1 x susceptible cv. Nannong 1138-2, we localized the gene mediating resistant to SMV-SC3 strain to a 90 kb interval on chromosome 2 in Kefeng No.1. Bean pod mottle virus (BPMV)-induced gene silencing (VIGS) were used to study the gene function of candidate genes in the mapping interval and revealed that an recombinant gene, later named as Rsc3K, caused by internal deletion of a genomic DNA frag ement in Kefeng No.1, is the resistant gene to SMV-SC3. By shuffling genes between avirulent isolate SC3 and avirulent SMV isolate 1129, we found that P3 is the virulence determinant causing resistance on Kefeng No.1. We showed the interaction between Rsc3K and P3 by the yeast two-hybrid (Y2H) and bimolecular fluorescent complementation (BiFC) assays. In conclusion, this study demonstrated that Rsc3K plays a crucial role in resistance of Kefeng No.1 to SMV-SC3 by direct interaction with viral protein P3.

HighlightRsc3K resistant to SMV-SC3 by interacting with P3.


title: Plasma membrane phosphatidylinositol-4-phosphate is not necessary for Candida albicans viability, yet is key for cell wall integrity and systemic infection

abstract:
Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi is required for the budding to filamentous growth transition in the human pathogenic fungus Candida albicans, however the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e. Efr3, Ypp1 and Stt4. The amount of plasma membrane PI(4)P in the efr3{Delta}/{Delta} and ypp1{Delta}/{Delta} mutant was [~]60% and [~]40% of the wild-type strain, respectively, whereas it was nearly undetectable in the stt4{Delta}/{Delta} mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity and had an increased exposure of cell wall {beta}(1,3)-glucan, yet they induced a range of hyphal specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3{Delta}/{Delta} had wild-type virulence, the ypp1{Delta}/{Delta} mutant had attenuated virulence and the stt4{Delta}/{Delta} mutant caused no lethality. In the mouse model of orpharyngeal candidiasis, only the ypp1{Delta}/{Delta} mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity and virulence in C. albicans.

ImportanceWhile the PI-4-kinases Pik1 and Stt4 both produce PI(4)P, the former generates PI(4)P at the Golgi and the latter at the plasma membrane and these two pools are functionally distinct. To address the importance of plasma membrane PI(4)P in Candida albicans, we have generated deletion mutants of the three putative plasma membrane PI-4-kinase complex components and quantified the levels of plasma membrane PI(4)P in each of these strains. Our work reveals that this phosphatidylinositol phosphate is specifically critical for the yeast-to-hyphal transition, cell wall integrity and virulence in a mouse systemic infection model. The significance of this work is in identifying a plasma membrane phospholipid that has an infection specific role, which is attributed to the loss of plasma membrane PI(4)P resulting in {beta}(1,3)-glucan unmasking.


title: High affinity enhancer-promoter interactions can bypass CTCF/cohesin-mediated insulation and contribute to phenotypic robustness

abstract: Transcriptional control by distal enhancers is an integral feature of gene regulation. To understand how enhancer-promoter interactions arise and assess the impact of disrupting 3D chromatin structure on gene expression, we generated an allelic series of mouse mutants that perturb the physical structure of the Sox2 locus. We show that in the epiblast and in neuronal tissues, CTCF-mediated loops are neither required for the interaction of the Sox2 promoter with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs between Sox2 and its distal enhancers generated ectopic loops with varying degrees of insulation that directly correlated with reduced transcriptional output. Yet, even the mutants exhibiting the strongest insulation, with six CTCF motifs in divergent orientation, could not fully abolish activation by distal enhancers, and failed to disrupt implantation and neurogenesis. In contrast, cells of the anterior foregut were more susceptible to chromatin structure disruption with no detectable SOX2 expression in mutants with the strongest CTCF-mediated boundaries. These animals phenocopied loss of SOX2 in the anterior foregut, failed to separate trachea from esophagus and died perinatally. We propose that baseline transcription levels and enhancer density may influence the tissue-specific ability of distal enhancers to overcome physical barriers and maintain faithful gene expression. Our work suggests that high affinity enhancer-promoter interactions that can overcome chromosomal structural perturbations, play an essential role in maintaining phenotypic robustness.


title: Seasonal challenges of tropical bats (Rousettus aegyptiacus) in temperate zones

abstract: Egyptian fruit bats (Rousettus aegyptiacus) manage to survive and flourish in a large geographic range despite the variability of natural and anthropogenic conditions in this range. To examine the challenges faced by free-ranging R.aegyptiacus living at the northern edge of their distribution, we performed a retrospective analysis of [~]1500 clinical cases reported by a bat rescue NGO over 25 months, from all over Israel. All cases of injured or stranded bats were evaluated and categorized according to date, place, sex, age, and etiology of the morbidity. The analysis of the data showed an increase in all types of morbidity during the wintertime, with more than twice the number of cases in comparison with the summertime, over two consecutive years. Moreover, we found that the number of abandoned pups peaks during spring till autumn when adult morbidity is minimal. We characterize two prominent types of previously undescribed morbidity in R.aegyptiacus, one in the form of bacterial illness, and the other associated with feet deformation which affects bats in addition to major anthropogenic-related threats related to synanthropic predators. We analyze the reasons driving winter morbidity and conclude that winter weather and specifically low temperature best explains this morbidity. We hypothesize that R.aegyptiacus, a fruit-bat of tropical origin is facing major seasonal difficulties near the northern edge of its distribution, probably limiting its further spread northward.

category: ecology


title: Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae

abstract: Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defence compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

category: evolutionary biology