title: Insights into the mutation T1117I in the spike and the lineage B.1.1.389 of SARS-CoV-2 circulating in Costa Rica

abstract:
Emerging mutations and genotypes of the SARS-CoV-2 virus, responsible for the COVID-19 pandemic, have been reported globally. In Costa Rica during the year 2020, a predominant genotype carrying the mutation T1117I in the spike (S:T1117I) was previously identified. To investigate the possible effects of this mutation on the function of the spike, i.e. the biology of the virus, different bioinformatic pipelines based on phylogeny, natural selection and co-evolutionary models, molecular docking and epitopes prediction were implemented.

Results of the phylogeny of sequences carrying the S:T1117I worldwide showed a polyphyletic group, with the emergency of local lineages. In Costa Rica, the mutation is found in the lineage B.1.1.389 and it is suggested to be a product of positive/adaptive selection. Different changes in the function of the spike protein and more stable interaction with a ligand (nelfinavir drug) were found. Only one epitope out 742 in the spike was affected by the mutation, with some different properties, but suggesting scarce changes in the immune response and no influence on the vaccine effectiveness.

Jointly, these results suggest a partial benefit of the mutation for the spread of the virus with this genotype during the year 2020 in Costa Rica, although possibly not strong enough with the introduction of new lineages during early 2021 which became predominant later. In addition, the bioinformatics pipeline offers an integrative and exhaustive in silico strategy to eventually study other mutations of interest for the SARS-CoV-2 virus and other pathogens.

HighlightsO_LIIn Costa Rica during the year 2020, a predominant SARS-CoV-2 genotype carrying the mutation T1117I in the spike (S:T1117I) was identified.
C_LIO_LIThe S:T1117I was assessed for possible effects of this mutation on the function of the spike with a in silico approach.
C_LIO_LIPhylogeny revealed that sequences carrying the S:T1117I worldwide define a polyphyletic group, with the emergency of local lineages, including the lineage B.1.1.389 in Costa Rica.
C_LIO_LIA positive/adaptive selection was identified for S:T1117I, with different changes in the function of the spike protein, more stable interaction with ligands and scarce changes in the immune response.
C_LIO_LIThe bioinformatics pipeline can be eventually used to study other mutations of the SARS-CoV-2 virus and other pathogens.
C_LI


title: Structure of a germline-like human antibody defines a neutralizing epitope on the SARS-CoV-2 spike NTD

abstract: Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germline-like monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against SARS-CoV-2 and determined its structure in complex with the spike glycoprotein by cryo-EM. We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its HCDR3 loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that - because of the conserved nature of the epitope - the mAb maintains binding to viral variants B.1.1.7 and B.1.351. Our study illustrates the feasibility of targeting the NTD to achieve broad neutralization against SARS-CoV-2 variants.

category: immunology


title: In Silico Defined SARS-CoV2 Epitopes May Not Predict Immunogenicity to COVID19

abstract: SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally, if not more important than humoral responses in mediating recovery and immune-protection. One of the major challenges in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities and consensus data. Our studies find that, in contrast to current dogma,  immunodominant SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes.


title: Antibody and T cell memory immune response after two doses of the BNT162b2 mRNA vaccine in older adults with and without prior SARS-CoV-2 infection

abstract: We quantified S1-specific IgG, neutralizing antibody titers, specific IFN{gamma} secreting T cells and functionality of specific CD4+ and CD8+ T cells in 130 young adults (median age 44.0 years) and 106 older residents living in a long-term care facility (86.5 years) after 2 doses of BNT162b2. Three months after the first injection, humoral and cellular memory responses were dramatically impaired in the 54 COVID-19-naive older compared to the 121 COVID-19-naive younger adults. Notably, older participants neutralizing antibodies, detected in 76.5% (versus 100% in young adults, P < 0.0001), were ten times lower than the youngers antibody titers (P < 0.0001). Antibody and T cell responses were greater among the 52 COVID-19-recovered than among the 54 COVID-19-naive older adults (P < 0.0001). Our study shows that 2 doses of BNT162b2 does not guarantee long-term protection against SARS-CoV-2 in the older. An additional dose should be considered to boost their specific memory response.


title: Phylogenomics Suggests the Origin of Obligately Anaerobic Anammox Bacteria Correlates with the Great Oxidation Event

abstract: The anaerobic ammonium oxidation (anammox) bacteria could transform ammonium and nitrite to dinitrogen gas, and this obligate anaerobic process accounts for up to half of the global nitrogen loss in surface environments. Yet its origin and evolution, which may give important insights into the biogeochemistry of early Earth, remains enigmatic. Here, we performed comprehensive phylogenomic analysis and showed a single origin of anammox bacteria within the phylum Planctomycetes. After accommodating the uncertainties and factors influencing time estimates, which includes implementing both a traditional cyanobacteria-based and a recently developed mitochondria-based approach, we estimated that anammox bacteria originated at early Proterozoic and most likely around the so-called Great Oxidation Event (GOE; 2.32 to 2.5 billion years ago [Ga]) which fundamentally changed global biogeochemical cycles. We further showed that during the origin of anammox bacteria, genes involved in oxidative stress, bioenergetics and anammox granules formation were recruited, which might have contributed to their survival on an increasingly oxic Earth. Our findings suggest the rising levels of atmospheric oxygen, which made nitrite increasingly available, was a potential driving force for the emergence of anammox bacteria. This is one of the first studies that link the GOE to the evolution of obligate anaerobic bacteria.

category: microbiology


title: Phylogenomic evidence for the Origin of Obligately Anaerobic Anammox Bacteria around the Great Oxidation Event


title: Citizen-science surveillance of triazole-resistant Aspergillus fumigatus in UK residential garden soils

abstract:
Compost is an ecological niche for Aspergillus fumigatus due to its role as a decomposer of organic matter and its ability to survive the high temperatures associated with the composting process. Subsequently, composting facilities are associated with high levels of A. fumigatus spores that are aerosolised from compost and cause respiratory illness in workers. In the UK, gardening is an activity enjoyed by individuals of all ages and it is likely that they are being exposed to A. fumigatus spores when handling commercial compost or compost they have produced themselves. In this study, 246 citizen scientists collected 509 soil samples from locations in their garden in the UK, from which were cultured 5,174 A. fumigatus isolates. Of these isolates, 736 (14%) were resistant to tebuconazole: the third most-sprayed triazole fungicide in the UK, which confers cross-resistance to the medical triazoles used to treat A. fumigatus lung infections in humans. These isolates were found to contain the common resistance mechanisms in the A. fumigatus cyp51A gene TR34/L98H or TR46/Y121F/T289A, and less common resistance mechanisms TR34, TR53, TR46/Y121F/T289A/S363P/I364V/G448S and (TR46)2/Y121F/M172I/T289A/G448S. Regression analyses found that soil samples containing compost were significantly more likely to grow susceptible and tebuconazole-resistant A. fumigatus than those that did not, and that compost samples grew significantly higher numbers of A. fumigatus than other samples.

ImportanceThese findings highlight compost as a potential health hazard to individuals with pre-disposing factors to A. fumigatus lung infections, and a potential health hazard to immunocompetent individuals who could be exposed to sufficiently high numbers of spores to develop infection. This raises the question of whether compost bags should carry additional health warnings regarding inhalation of A. fumigatus spores, whether individuals should be advised to wear facemasks whilst handling compost or whether commercial producers should be responsible for sterilising compost before shipping. The findings support increasing public awareness of the hazard posed by compost and investigating measures that can be taken to reduce the exposure risk.


title: Kinetic characterization and thermostability of C. elegans cytoplasmic and mitochondrial malate dehydrogenases

abstract: Malate dehydrogenase (MDH) catalyzes the conversion of NAD+ and malate to NADH and oxaloacetate in the last step of the citric acid cycle. Eukaryotes have at least two MDH isozymes, one that is imported into the mitochondria and one that remains in the cytoplasm. We overexpressed and purified Caenorhabditis elegans cytoplasmic MDH-1 (F46E10.10) and mitochondrial MDH-2 (F20H11.3) in E. coli. Our goal was to compare the kinetic and structural properties of these enzymes because C. elegans can survive adverse environmental conditions, such as lack of food and elevated temperatures. In steady-state enzyme kinetics assays, we determined that the KM values for oxaloacetate were 54 and 52 M, and the KM values for NADH were 61 and 107 M, for MDH-1 and MDH-2, respectively. We partially purified endogenous MDH from a mixed population of worms and separated MDH-1 from MDH-2 using anion exchange chromatography. Both endogenous enzymes had a KM for oxaloacetate similar to that of the corresponding recombinant enzyme. The reaction velocities of the recombinant enzymes had slightly different temperature-dependencies: MDH-1 and MDH-2 had maximum activity at 40 {degrees}C and 35 {degrees}C, respectively. In a thermotolerance assay, MDH-1 was much more thermostable than MDH-2. Molecular homology modeling predicted that MDH-1 had more salt-bridges between the subunits than mammalian MDH1 enzymes, and these ionic interactions may contribute to its thermostability. In contrast, the MDH-2 homology model predicted fewer ionic interaction between the subunits compared to mammalian MDH2 enzymes. These results suggest that the increased structural stability of MDH-1 may facilitate its ability to remain active in adverse environmental conditions. In contrast, MDH-2 may use other strategies, such as protein binding partners, to function under similar conditions.

category: biochemistry


title: MLKL D144K mutation activates the necroptotic activity of the N-terminal MLKL domain

abstract: Mixed-lineage kinase domain-like protein (MLKL) is an essential effector protein of necroptotic cell death. The four-helix bundle domain (4HB) presented by the first 125 amino acids of the N-terminal domain is sufficient for its necroptotic activity. However, it has been proposed that the subsequent helix H6 of the brace region has a regulatory effect on its necroptotic activity. How the brace region restrains the necroptotic activity of the N-terminal domain of MLKL is currently unknown. Here, we demonstrate the importance of helix H6 to constrain the necroptotic activity. A single amino acid mutation D144K was able to activate the necroptotic activity of the N-terminal domain of MLKL by removing helix H6 away from 4HB domain. This enabled proteins oligomerization and membrane translocation. Moreover, a biophysical comparison revealed that helix H6 becomes partially unstructured due to D144K mutation, leading to a lower overall thermodynamic stability of the mutant protein compared to the wild type.


title: Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development

abstract: Fungal infection is an important public health problem afflicting more than a billion people worldwide. Mycoses are especially important in Latin America, and in Brazil in particular. Paracoccidioides is the genus of fungi responsible for paracoccidioidomycosis comprising two species, P. brasiliensis and P. lutzii. The lungs are the primary infection site, but oral mucosa and airways can also be affected. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells and is the most studied protein of P. brasiliensis. Seminal work identified a specific stretch of 15 amino acids that spans the region 181-195 (called P10) as an important epitope of gp43, being recognized by T lymphocytes in peripheral blood mononuclear cells of mice and humans and is envisioned as a potential vaccine component. Here, we show by using thioflavin T (ThT), transmission electron microscopy and other methods that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. In silico, aggregation analysis reveals several aggregation-prone regions (APR) in the P10 sequence that are capable of forming amyloid cores with steric zipper architecture. Seeds of P10 obtained by fibril mechanical fragmentation were able to induce the aggregation of P4 but not P23, as evidenced by ThT binding and mass spectrometry. These two peptides, also derived from gp43, are potent modulators of local and systemic inflammation. In-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by the action of several proteases such as proteinase K, trypsin and pepsin, which suggests that P10 could be formed upon gp43 digestion in a physiological condition. Considering our data in the context of a potential vaccine development, we redesigned the sequence of the P10 peptide, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.


title: Atomistic Simulations and Deep Mutational Scanning of Protein Stability and Binding Interactions in the SARS-CoV-2 Spike Protein Complexes with Nanobodies: Molecular Determinants of Mutational Escape Mechanisms

abstract: Structural and biochemical studies have recently revealed a range of rationally engineered nanobodies with efficient neutralizing capacity against SARS-CoV-2 virus and resilience against mutational escape. In this work, we combined atomistic simulations and conformational dynamics analysis with the ensemble-based mutational profiling of binding interactions for a diverse panel of SARS-CoV-2 spike complexes with nanobodies. Using this computational toolkit we identified dynamic signatures and binding affinity fingerprints for the SARS-CoV-2 spike protein complexes with nanobodies Nb6 and Nb20, VHH E, a pair combination VHH E+U, a biparatopic nanobody VHH VE, and a combination of CC12.3 antibody and VHH V/W nanobodies. Through ensemble-based deep mutational profiling of stability and binding affinities, we identify critical hotspots and characterize molecular mechanisms of SARS-CoV-2 spike protein binding with single ultra-potent nanobodies, nanobody cocktails and biparatopic nanobodies. By quantifying dynamic and energetic determinants of the SARS-CoV-2 S binding with nanobodies, we also examine the effects of circulating variants and escaping mutations. We found that mutational escape mechanisms may be controlled through structurally and energetically adaptable binding hotspots located in the host receptor-accessible binding epitope that are dynamically coupled to the stability centers in the distant epitope targeted by VHH U/V/W nanobodies. The results of this study suggested a mechanism in which through cooperative dynamic changes, nanobody combinations and biparatopic nanobody can modulate the global protein response and induce the increased resilience to common escape mutants.

category: biophysics


title: Differential effects of high versus low linear energy transfer (LET) radiation on type-I interferon (IFNβ) and TREX1 responses

abstract:
PurposeCancer cells produce innate immune signals following radiation damage, with STING pathway signaling as a critical mediator. High linear energy transfer (LET) radiations create larger numbers of DNA double-strand breaks (DSBs) per unit dose than low-LET radiations and may therefore be more immunogenic. We studied the dose response characteristics of pro-immunogenic type-I interferon, interferon-beta (IFN{beta}), and its reported suppressor signal, three-prime repair exonuclease 1 (TREX1), in vitro with low-LET x-rays and high-LET fast neutrons.

MethodsMerkel cell carcinoma cells (MCC) were irradiated by graded doses of x-rays (1-24 Gy) or fast neutrons (1-8 Gy). IFN{beta} was measured as a function of dose via ELISA assay, and exonuclease TREX1 expression via immunofluorescence microscopy. The Monte Carlo damage simulation (MCDS) was used to model fast neutron relative biological effectiveness for DSB induction (RBEDSB) and compared to laboratory measurements of the RBE for IFN{beta} production (RBEIFN{beta}) and TREX1 upregulation (RBETREX1). RBEIFN{beta} models were also applied to radiation transport simulations to quantify the potential secretion of IFN{beta} in representative clinical beams.

ResultsPeak IFN{beta} secretion occurred at 5.7 Gy for fast neutrons and at 14.0 Gy for x-rays, i.e., an effective RBEIFN{beta} of 2.5 {+/-} 0.2. The amplitude (peak value) of secreted IFN{beta} signal did not significantly differ between x-rays and fast neutrons (P > 0.05). TREX1 signal increased linearly with absorbed dose, with a four-fold higher upregulation per unit dose for fast neutrons relative to x-rays (RBETREX1 of 4.0 {+/-} 0.1). Monte Carlo modeling of IFN{beta} suggests Bragg peak-to-entrance ratios of IFN{beta} production of 40, 100, and 120 for proton, alpha, and carbon ion beams, respectively, a factor of 10-20-fold higher compared to their corresponding physical dose peak-to-entrance ratios. The spatial width of the Bragg peak for IFN{beta} production is also a factor of two smaller.

ConclusionHigh-LET fast neutrons initiate a larger IFN{beta} response per unit absorbed dose than low-LET x-rays (i.e., RBEIFN{beta} value of 2.5). The RBE value for IFN{beta} is quite similar to data reported in the literature for DSB induction and cellular, post-irradiation micronucleation formation for neutrons and x-rays. The increased IFN{beta} release after high-LET radiation may be a contributing factor in stimulating a systemic anti-tumor, adaptive immune response (abscopal effect). However, our results indicate that TREX1 anti-inflammatory signaling in vitro for MCC cells is larger per unit dose for fast neutrons than for x-rays (RBETREX1 of 4.0). Given these competing effects, additional studies are needed to clarify whether or not high-LET radiations are therapeutically advantageous over low-LET radiation for pro-inflammatory immune signaling in other cell lines in vitro and for in vivo cancer models.

category: cancer biology


title: Anti-tumor efficacy of an MMAE conjugated antibody targeting cell surface TACE/ADAM17-cleaved Amphiregulin in breast cancer

abstract: The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we report the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection.


title: Evaluation of intranasal nafamostat or camostat for SARS-CoV-2 chemoprophylaxis in Syrian golden hamsters

abstract: Successful development of a chemoprophylaxis against SARS-CoV-2 could provide a tool for infection prevention implementable alongside vaccination programmes. Camostat and nafamostat are serine protease inhibitors that inhibit SARS-CoV-2 viral entry in vitro but have not been characterised for chemoprophylaxis in animal models. Clinically, nafamostat is limited to intravenous delivery and while camostat is orally available, both drugs have extremely short plasma half-lives. This study sought to determine whether intranasal dosing at 5 mg/kg twice daily was able to prevent airborne transmission of SARS-CoV-2 from infected to uninfected Syrian golden hamsters. SARS-CoV-2 viral RNA was above the limits of quantification in both saline- and camostat-treated hamsters 5 days after cohabitation with a SARS-CoV-2 inoculated hamster. However, intranasal nafamostat-treated hamsters remained RNA negative for the full 7 days of cohabitation. Changes in body weight over the course of the experiment were supportive of a lack of clinical symptomology in nafamostat-treated but not saline- or camostat-treated animals. These data are strongly supportive of the utility of intranasally delivered nafamostat for prevention of SARS-CoV-2 infection and further studies are underway to confirm absence of pulmonary infection and pathological changes.

category: pharmacology and toxicology


title: Synthetic metabolic pathways for conversion of CO2 into secreted short- to medium-chain hydrocarbons using cyanobacteria

abstract:
The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of bioreaction ingredients it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the bioreaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioestersaes. When UndB instead was combined with a C12-specific  UcFatB1 thioesterase, this resulted in ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Optimization of the RBS, promoter and gene order in these synthetic operons resulted in 1-alkene bioproductivity of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was accomplished and optimal results were obtained with UndB and a C12 chain-length specific thioesterase.

HighlightsO_LIMultiple repression of endogenous aldehyde reductases/dehydrogenases by CRISPRi enabled propane biosynthesis
C_LIO_LIBiosynthesis of short-medium chain hydrocarbons (C7-C11) in a cyanobacterium was demonstrated for the first time
C_LIO_LIThe final enzymes of the hydrocarbon pathways influenced both productivity and product profile
C_LIO_LIAll volatile products were naturally secreted and accumulated outside of the cell
C_LI

category: synthetic biology


title: Intracellular phase separation of globular proteins facilitated by short cationic peptides

abstract:
Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We add to this toolbox by designing short, highly charged peptide tags that provide several key advantages for engineering protein phase separation. Herein, we report the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

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title: A natural marmoset model of genetic generalized epilepsy.

abstract:
As a common neurological disease, epilepsy has been extensively studied. Efforts have been made on rodent and other animal models to reveal the pathogenic mechanisms of epilepsy and develop new drugs as treatment. However, the features of current epilepsy models cannot fully mimic different kinds of epilepsy in human, asking for non-human primates models of epilepsy. The common marmoset (Callithrix jacchus) is a New World monkey that is widely used to study brain function. Here, we show a natural marmoset model of generalized epilepsy. In this unique marmoset family, generalized epilepsy was successfully induced by handling operation in some individuals. We mapped the marmoset family with handling-sensitive epilepsy and found that epileptic marmoset had an autosomal dominant genetic predisposition. Those marmosets were more sensitive to epilepsy inducers pentylenetetrazol (PTZ). By electrocorticogram (ECoG) recording, we detected epileptic discharge in marmoset with history of seizures. However, there was no significant change in the overall structure of epileptic marmoset brain. In summary, we report a family of marmosets with generalized seizures induced by handling operation. This epileptic marmoset family provides insights to better understand the mechanism of generalized epilepsy and helps to develop new therapeutic methods.

Short summaryDespite the rodent and other animal models on epilepsy, a common neurological disease, the understanding of the pathogenic mechanisms of epilepsy and treatment are yet to be advanced., Non-human primates are good experimental animals for modelling neurological disease. Here we report a common marmoset family with generalized epilepsy induced by handling stimulation. We mapped the epileptic marmoset family and found that epilepsy had a genetic predisposition. The present report deals in detail with the phenotypes and characteristics of epileptic marmosets. This natural marmoset reflect epilepsy model will help to better understand the mechanism of epilepsy and develop new therapeutic methods.

category: neuroscience


title: Nicotine dependence and functional connectivity of insular cortex subregions

abstract: The insular cortex has been identified as a promising target in brain-based therapies for Tobacco Use Disorder, and has three major sub-regions (ventral anterior, dorsal anterior, and posterior) that serve distinct functional networks. How these subregions and associated networks contribute to nicotine dependence has not been well understood, and therefore was the subject of this study. Forty-seven individuals (24 women; 18-45 years old) who smoked cigarettes daily rated their dependence using the Fagerstrom Test for Nicotine Dependence (FTND), abstained from smoking overnight (~12 h), and underwent resting-state functional MRI. Correlations between dependence and resting-state functional connectivity (RSFC) of the major insular sub-regions were evaluated using whole-brain-corrected voxel-wise analyses and post-hoc region-of-interest (ROI) analyses. Dependence was analyzed both as a unitary (FTND total score) and bivariate construct - two FTND factors ("morning smoking" and "daytime smoking"). Dependence was negatively correlated with connectivity of both the right dorsal and left ventral anterior insula with the left precuneus, and with connectivity of the left posterior insula to the left putamen. In post-hoc analyses, dependence correlated negatively with connectivity between all anterior insula subregions and the left precuneus, and with bilateral posterior insula connectivity with the left posterior putamen. The latter finding was driven by "daytime smoking". These results suggest an anterior-posterior distinction in functional insular networks associated with different dimensions of nicotine dependence, with greater dependence linked to weaker connectivity. They may inform therapeutic approaches involving brain stimulation that may elicit differential clinical outcomes depending on the insular subnetwork targeted.


title: Age-associated changes to neuronal dynamics involve a loss of inhibitory signaling in C. elegans

abstract: In the aging brain, many of the alterations underlying cognitive and behavioral decline remain opaque. C. elegans offers a powerful model for aging research, with a simple, well-studied nervous system to further our understanding of the cellular modifications and functional alterations accompanying senescence. We perform multi-neuronal functional imaging across the aged C. elegans nervous system, measuring an age-associated breakdown in system-wide functional organization. At single-cell resolution, we detect shifts in activity dynamics toward higher frequencies. In addition, we measure a specific loss of inhibitory signaling that occurs early in the aging process and alters the systems critical excitatory/inhibitory balance. These effects are recapitulated with mutation of the calcium channel subunit UNC-2/CaV2. We find that manipulation of inhibitory GABA signaling can partially ameliorate or accelerate the effects of aging. The effects of aging are also partially mitigated by disruption of the insulin signaling pathway, known to increase longevity, or by a reduction of caspase activation. Data from mammals are consistent with our findings, suggesting a conserved shift in the balance of excitatory/inhibitory signaling with age that leads to breakdown in global neuronal dynamics and functional decline.


title: Age-associated changes to neuronal dynamics involve a disruption of excitatory/inhibitory balance in C. elegans


title: Subspace alignment as a mechanism for binding

abstract: To choose between options, we must solve two important binding problems. First, the features that determine each options values must be appropriately combined and kept separate from the corresponding features of other options. Second, options must be associated with the specific actions needed to select them. We hypothesized that the brain solves these problems through use of aligned (for bound dimensions) and orthogonal (for separated dimensions) population subspaces. We examined responses of single neurons in six putative value-coding regions in rhesus macaques performing a risky choice task. In all areas, single neurons encode the features that define the value of each option (stakes and probability) but only very weakly encode value per se. However, the coding dimensions associated with these features are aligned on a single subspace, from which a strong emergent value signal can be read out. Moreover, all six regions use nearly orthogonal subspaces for the left and right options, thereby linking options to their position in space, implementing functional partitioning, and reducing the possibility of misbinding. These results provide a new solution to the neuroeconomic binding problems and suggest that other forms of binding may work through similar principles.


title: A realistic locomotory model of Drosophila larva for behavioral simulations.

abstract: The Drosophila larva is extensively used as model species in experiments where behavior is recorded via tracking equipment and evaluated via population-level metrics. Although larva locomotion neuromechanics have been studied in detail, no comprehensive model has been proposed for realistic simulations of foraging experiments directly comparable to tracked recordings. Here we present a virtual larva for simulating autonomous behavior, fitting empirical observations of spatial and temporal kinematics. We propose a trilayer behavior-based control architecture for larva foraging, allowing to accommodate increasingly complex behaviors. At the basic level, forward crawling and lateral bending are generated via coupled, interfering oscillatory processes under the control of an intermittency module, alternating between crawling bouts and pauses. Next, navigation in olfactory environments is achieved via active sensing and topdown modulation of bending dynamics by concentration changes. Finally, adaptation at the highest level entails associative learning. We could accurately reproduce behavioral experiments on autonomous free exploration, chemotaxis, and odor preference testing. Interindividual variability is preserved across virtual larva populations allowing for single animal and population studies. Our model is ideally suited to interface with neural circuit models of sensation, memory formation and retrieval, and spatial navigation.


title: Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer

abstract: The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T cell function and their relation to patient outcome. Here we leverage single-cell RNA-sequencing (scRNA-seq) analysis of several mouse and human tumors and find that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. Using mouse models, we also find that tumor monocyte-to-macrophage progression is profoundly tied to regulatory T cell (Treg) abundance. Importantly, in human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states in kidney tumors.


title: Pyroptosis-dependent and -independent cross-priming of CD8+ T cells by intestinal epithelial cell-derived antigen

abstract: The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens are cross-presented to CD8+ T cells. However, activation of CD8+ T cells by IEC-derived antigen only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (cDC1), whereas cross-priming in the presence of pyroptosis did not. These data suggest the existence of parallel pyroptosis-dependent and pyroptosis-independent but cDC1-dependent pathways for cross-presentation of IEC-derived antigens.


title: EpicPCR-Directed Cultivation of a Candidatus Saccharibacteria Symbiont Reveals a Type IV Pili-dependent Epibiotic Lifestyle

abstract: Candidate phyla radiations (CPR), accounting for a major microbial supergroup with remarkably small genomes and reduced sizes, are widely distributed yet mostly uncultured. Limited culture and its obligate reliance upon other bacteria hindered investigation of their lifestyles. In this work we isolated a CPR bacterium, TM7i, with its host Leucobacter aridocollis J1, by combination of Emulsion, Paired Isolation and Concatenation PCR (epicPCR) detection and filtrate co-culture. Genomic profiling of TM7 genomes and microscopic investigation of TM7i-J1 symbiosis suggest the conservation of type IV pili and a pili-dependent lifestyle of TM7. Further, we observed twitching motility of TM7i mediated by pili and its role played in the interaction with its host. Our results shed a light on the lifestyle about this enigmatic bacterial radiation, which may also be adopted by other CPR organisms. The epicPCR-directed isolation method underlines high efficiency of CPR bacteria isolation and thus may be used in other symbiotic or epibiotic microorganisms.


title: Founder cell configuration drives competitive outcome within colony biofilms

abstract: Bacteria typically form dense communities called biofilms, where cells are embedded in a self-produced extracellular matrix. Competitive interactions between strains within the biofilm context are studied due to their potential applications in biological, medical, and industrial systems. Combining mathematical modelling with experimental assays, we reveal that the spatial structure and the competitive dynamics within biofilms are significantly affected by the location and density of founder cells. Using an isogenic pair of Bacillus subtilis strains, we show that the observed spatial structure and relative strain biomass in a mature biofilm can be mapped directly to the locations of founder cells. Moreover, we define a predictor of competitive outcome that accurately forecasts relative abundance of strains based solely on the founder cells access to free space. Consequently, we reveal that variability of competitive outcome in biofilms inoculated at low founder density is a natural consequence of the random positioning of founding cells in the inoculum. Extending our study to non-isogenic strain pairs of B. subtilis, we show that even for strains with different antagonistic strengths, a race for space remains the dominant mode of competition in biofilms inoculated at low founder densities. Our results highlight the importance of spatial dynamics on competitive interactions within biofilms and hence to related applications.


title: Multifactorial role of mitochondria in echinocandin tolerance revealed by transcriptome analysis of drug-tolerant cells

abstract:
Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target, {beta}-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida, a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, the latter of which was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypo-tolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance.

ImportanceEchinocandin drugs are a first line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypo-tolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.


title: Combinations of Peptides Synergistically Activate the Regenerative Capacity of Skin Cells In Vitro

abstract:
OBJECTIVETo explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination in different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared to the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations.

METHODSMicroarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment.

RESULTSMicroarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared to individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS, and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, a second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism whereby peptide synergies may accelerate skin repair.

CONCLUSIONThrough combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.

category: molecular biology


title: Cytoplasmic Switch of ARS2 Isoforms Promotes Nonsense-Mediated mRNA Decay and Arsenic Sensitivity

abstract: The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2s intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1, ERF1 and DHX34, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, stress response, and cancer treatment.


title: Ultrastructural analysis of nasopharyngeal epithelial cells from patients with SARS-CoV-2 infection

abstract: The nasal epithelium is an initial site for SARS-CoV-2 infection, responsible for the ongoing COVID-19 pandemic. However, the pathogenicity and morphological impact of SARS-CoV-2 on the nasopharynx cells from symptomatic patients with different viral loads remain poorly understood. Here, we investigated the ultrastructure of nasal cells obtained from individuals at distinct disease days and with high and low SARS-CoV-2 loads. Squamous and ciliated cells were the main cells observed in SARS-CoV-2 negative samples. We identified virus-like particles (VLPs) and replication organelles (RO)-like structures in the squamous cells from high viral load samples after 3- and 4-days of symptoms. Ultrastructural changes were found in those cells, such as the loss of microvilli and primary cilium, the increase of multivesicular bodies and autophagosomes, and signs of cell death. No ciliated cells were found in those samples. Squamous cells from low viral load sample after 5 days of symptoms showed few microvilli and no primary cilium. VLPs and RO-like structures were found in the ciliated cells only. No ultrastructural alterations were seen in the cells from low viral load individuals after 10- and 14-days of symptoms. Our results shed light on the ultrastructural effects of SARS-CoV-2 infection on the human nasopharyngeal cells.

category: cell biology


title: A single cell transcriptome atlas reveals the heterogeneity of the healthy human cornea and identifies novel markers of the corneal limbus and stroma

abstract: The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowmans layer, stroma, Descemets membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified that the expression of CAV1, CXCL14, HOMER3 and CPVL were exclusive to the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.


title: The EGF-motif containing protein SPE-36 is a secreted protein required for sperm function at fertilization in C. elegans

abstract: The growing number of genes specifically required for fertilization suggests that there is a significant amount of molecular complexity at the sperm-egg interface. Thus, we have adopted a model of a "fertilization synapse" where specialized zones of interaction and multi-protein complexes mediate gamete interaction and fusion. The fertilization synapse is likely to be composed of both trans and cis protein-protein interactions at the surface of each gamete. Mutations in the Caenorhabditis elegans spe-36 gene result in a sperm-specific fertility defect. Surprisingly, spe-36 encodes a secreted EGF-motif containing protein that functions cell autonomously. Despite the fact that morphology and migratory behavior of spe-36 sperm are indistinguishable from wild-type sperm, spe-36 sperm make close contact with oocytes but fail to fertilize them. The genetic requirement for a secreted sperm-derived protein for fertilization is novel and represents a paradigm-shifting discovery in the molecular understanding of fertilization.

category: developmental biology


title: Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF

abstract: The eukaryotic genome is tightly packed inside the nucleus, where it is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors that bind DNA, such as the multi-zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but the impact of such structure on genome function during the first stages of mammalian development is still unclear. Here, we show that deletion of the Ctcf gene in mouse embryos impairs the correct establishment of chromatin structure, but initial lineage decisions take place and embryos are viable until the late blastocyst stage. Furthermore, we observe that maternal CTCF is not necessary for development. Transcriptomic analyses of mutant embryos show that the changes in metabolic and protein homeostasis programs that occur during the progression from the morula to the blastocyst depend on CTCF. Yet, these changes in gene expression do not correlate with disruption of chromatin structure, but mainly with proximal binding of CTCF to the promoter region of genes downregulated in mutants. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but mostly as independent processes.


title: SPE-51, a sperm secreted protein with an Immunoglobulin-like domain, is required for sperm-egg fusion in C. elegans

abstract: Despite the importance of fertilization, the molecular basis of sperm-egg interaction is not well understood. In a forward genetics screen for fertility mutants in Caenorhabditis elegans we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell-autonomously and localizes to the surface of the spermatozoa. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.


title: Concurrent evolution of anti-aging gene duplications and cellular phenotypes in long-lived turtles

abstract: There are many costs associated with increased body size and longevity in animals, including the accumulation of genotoxic and cytotoxic damage that comes with having more cells and living longer. Yet, some species have overcome these barriers and have evolved remarkably large body sizes and long lifespans, sometimes within a narrow window of evolutionary time. Here, we demonstrate through phylogenetic comparative analysis that multiple turtle lineages, including Galapagos giant tortoises, concurrently evolved large bodies, long lifespans, and reduced cancer risk. We also show through comparative genomic analysis that Galapagos giant tortoises have gene duplications related to longevity and tumor suppression. To examine the molecular basis underlying increased body size and lifespan in turtles, we treated cell lines from multiple species, including Galapagos giant tortoises, with drugs that induce different types of cytotoxic stress. Our results indicate that turtle cells, in general, are resistant to oxidative stress related to aging, while Galapagos giant tortoise cells, specifically, are sensitive to endoplasmic reticulum stress, which may give this species an ability to mitigate the effects of cellular stress associated with increased body size and longevity.

category: evolutionary biology


title: Growth temperature is the principal driver of chromatinization in archaea

abstract: Across the tree of life, DNA in living cells is associated with proteins that coat chromosomes, constrain their structure and influence DNA-templated processes such as transcription and replication. In bacteria and eukaryotes, HU and histones, respectively, are the principal constituents of chromatin, with few exceptions. Archaea, in contrast, have more diverse repertoires of nucleoid-associated proteins (NAPs). The evolutionary and ecological drivers behind this diversity are poorly understood. Here, we combine a systematic phylogenomic survey of known and predicted NAPs with quantitative protein abundance data to shed light on the forces governing the evolution of archaeal chromatin. Our survey highlights the Diaforarchaea as a hotbed of NAP innovation and turnover. Loss of histones and Alba in the ancestor of this clade was followed by multiple lineage-specific horizontal acquisitions of DNA-binding proteins from other prokaryotes. Intriguingly, we find that one family of Diaforarchaea, the Methanomethylophilaceae, lacks any known NAPs. Comparative analysis of quantitative proteomics data across a panel of 19 archaea revealed that investment in NAP production varies over two orders of magnitude, from <0.02% to >5% of total protein. Integrating genomic and ecological data, we demonstrate that growth temperature is an excellent predictor of relative NAP investment across archaea. Our results suggest that high levels of chromatinization have evolved as a mechanism to prevent uncontrolled helix opening and runaway denaturation - rather than, for example, to globally orchestrate gene expression - with implications for the origin of chromatin in both archaea and eukaryotes.


title: Fully resolved assembly of Cryptosporidium parvum

abstract:
BackgroundCryptosporidium parvum is an apicomplexan parasite commonly found across many host species with a global infection prevalence in human populations of 7.6%. As such, it is important to understand the diversity and genomic makeup of this prevalent parasite to fight established infections and prohibit further transmission. The basis of every genomic study is a high quality reference genome that has continuity and completeness, thus enabling comprehensive comparative studies.

FindingsHere, we provide a highly accurate and complete reference genome of Cryptosporidium parvum. The assembly is based on Oxford Nanopore reads and was improved using Illumina reads for error correction. We also outline how to evaluate and choose from different assembly methods based on two main approaches that can be applied to other Cryptosporidium species. The assembly encompasses 8 chromosomes and includes 13 telomeres that were resolved. Overall, the assembly shows a high completion rate with 98.4% single copy BUSCO genes.

ConclusionsThis high quality reference genome of a zoonotic IIaA17G2R1 C. parvum subtype isolate provides the basis for subsequent comparative genomic studies across the Cryptosporidium clade. This will enable improved understanding of diversity, functional and association studies.

category: genomics


title: Protein kinase signalling at the Leishmania kinetochore captured by XL-BioID

abstract: Elucidating protein kinase signaling pathways is an important but challenging problem in cell biology. Phosphoproteomics has been used to identify many phosphorylation sites, however the spatial context of these sites within the cell is mostly unknown, making it difficult to reconstruct signalling pathways. To address this problem an in vivo proximity capturing workflow was developed, consisting of proximity biotinylation followed by protein cross-linking (XL-BioID). This was applied to protein kinases of the Leishmania kinetochore, leading to the discovery of a novel essential kinetochore protein, KKT26. XL-BioID enabled the quantification of proximal phosphosites at the kinetochore through the cell cycle, allowing the phosphorylation state of the kinetochore to be followed during assembly. A specific inhibitor of kinetochore protein kinases KKT10/KKT19 was used to show that XL-BioID provides a spatially focussed view of protein kinase inhibition, identifying 16 inhibitor-responsive proximal phosphosites, including 3 on KKT2, demonstrating the potential of this approach for discovery of in vivo kinase signalling pathways.


title: The multilayered organization of water-soluble proteins

abstract:
The structural analysis of proteins has focused primarily on secondary structure, three-dimensional fold and active site while whole surface has been analyzed to a lesser extent and interior has not received much attention. Here we present an analysis of both the surfaces and the interiors of a set of water-soluble monomeric proteins in terms of solvent-excluded surface (SES) and atomic partial charge. The analysis shows that the surface of a soluble monomer has a net negative charge and is much smoother than the interior. Most interestingly with regard to both atomic partial charge and SES-defined geometric property there exists a multilayered organization from the exterior to the interior of a soluble monomer. The multilayered organization is closely related to protein-solvent interaction and should be a general feature of a water-soluble protein. Particularly the multilayered organization may set an upper limit for the size of a water-soluble monomer and plays an important role in the determination of its overall shape in solution.

Significance statementThe analysis of the solvent-excluded surfaces (SESs) of a large set of water-soluble monomers with crystal structures shows that in any soluble monomer there exists a multilayered organization in terms of SES-defined electric and geometric properties. It means that the atoms in a soluble monomer are not randomly distributed but organized into successive layers to optimize its interaction with solvent molecules. The SES-derived multi-layered organization should be a general feature of a water-soluble protein and likely plays a vital role in its solvation, folding and structure.


title: Pseudomonas syringae effector AvrE associates with plant membrane nanodomains and binds phosphatidylinositides in vitro

abstract: Bacterial phytopathogens deliver effector proteins into host cells as key virulence weapons to cause disease. Extensive studies revealed diverse functions and biochemical properties of different effector proteins from pathogens. In this study, we show that the Pseudomonas syringae effector AvrE, the founding member of a broadly conserved and pathologically important bacterial effector family, binds to phosphatidylinositides (PIPs) in vitro and shares some properties with eukaryotic PROPPINs ({beta}-propellers that bind polyphosphoinositides). In planta pull down experiments with transgenic Arabidopsis plants expressing AvrE revealed that AvrE is associated with several plant proteins including plasma membrane lipid-raft proteins. These results shed new light on the properties of a bacterial effector that is crucial for bacterial virulence in plants.

category: pathology


title: The role of posterior ventral stream areas for viewpoint-invariant object recognition

abstract: Object constancy is one of the most crucial mechanisms of the human visual system enabling viewpoint invariant object recognition. However, the neuronal foundations of object constancy are widely unknown. Research has shown that the ventral visual stream is involved in processing of various kinds of object stimuli and that several regions along the ventral stream are possibly sensitive to the orientation of an object in space. To systematically address the question of viewpoint sensitive object perception, we conducted a study with stroke patients as well as an fMRI experiment with healthy participants applying object stimuli in several spatial orientations, for example in typical and atypical viewing conditions. In the fMRI experiment, we found stronger BOLD signals and above-chance classification accuracies for objects presented in atypical viewing conditions in fusiform face sensitive and lateral occipito-temporal object preferring areas. In the behavioral patient study, we observed that lesions of the right fusiform gyrus were associated with lower performance in object recognition for atypical views. The complementary results from both experiments emphasize the contributions of fusiform and lateral-occipital areas to visual object constancy and indicate that visual object constancy is particularly enabled through increased neuronal activity and specific activation patterns for objects in demanding viewing conditions.


title: CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

abstract: Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.


title: Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo

abstract: The human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least 8 previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by complex mutational processes, including multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo.


title: Positive selection and enhancer evolution shaped lifespan and body mass in great apes

abstract: Within primates, the great apes are outliers both in terms of body size and lifespan, since they include the largest and longest-lived species in the order. Yet, the molecular bases underlying such features are poorly understood. Here, we leveraged an integrated approach to investigate multiple sources of molecular variation across primates, focusing on ~1,550 genes previously described as tumor suppressors, oncogenes, ageing genes in addition to a novel Build of the CellAge database of cell-senescence genes (version 2), herein presented for the first time. Specifically, we analyzed dN/dS rates, positive selection, gene expression (RNA-seq) and gene regulation (ChIP-seq). By analyzing the correlation between dN/dS, maximum lifespan and body mass we identified 67 genes that in primates co-evolved with those traits. Further, we identified 6 genes, important for immunity, neurodevelopment and telomere maintenance (including TERF2), under positive selection in the great ape ancestor. RNA-seq data, generated from the liver of six species representing all the primate lineages, revealed that ~8% of the longevity genes are differentially expressed in apes relative to other primates. Importantly, by integrating RNA-seq with ChIP-seq for H3K27ac (which marks active enhancers), we show that the differentially expressed longevity genes are significantly more likely than expected to be located near a novel "ape-specific" enhancer. Moreover, these particular ape-specific enhancers are enriched for young transposable elements, and specifically SINE-Vntr-Alus (SVAs). In summary, we demonstrate that multiple evolutionary forces have contributed to the evolution of lifespan and body size in primates.


title: Allele-specific expression links diverse cis-regulatory mutations to recurrent gene dysregulation in high-risk neuroblastoma

abstract: Neuroblastoma is a pediatric malignancy with a high frequency of metastatic disease at initial diagnosis. Neuroblastoma tumors have few protein-coding mutations but contain extensive somatic copy number alterations (SCNAs) suggesting that mutations that alter gene dosage are important drivers of tumorigenesis. Here we analyze allele-specific expression (ASE) in 96 high-risk neuroblastoma tumors to discover genes with cis-acting mutations that alter dosage. We identify 1,049 genes with recurrent, neuroblastoma-specific ASE, 68% of which lie within common SCNA regions. However, many genes exhibit ASE in copy neutral samples and are enriched for mutations that cause nonsense-mediated decay, indicating that neuroblastoma tumors select for multiple types of mutations that alter gene expression. We also find 24 genes with reduced expression in stage 4 disease that have neuroblastoma-specific ASE that is independent of SCNAs. At least two of these genes have evidence for tumor suppressor activity including the transcription factor TFAP2B and the protein tyrosine phosphatase PTPRH. In summary, our ASE analysis discovers genes that are recurrently dysregulated by both large SCNAs and other cis-acting mutations in high-risk neuroblastoma.


title: The geometry of domain-general performance monitoring representations in the human medial frontal cortex

abstract: Controlling behavior to flexibly achieve desired goals depends on the ability to monitor ones own performance. It is unknown how performance monitoring can be both flexible to support different tasks and specialized to perform well on each. We recorded single neurons in the human medial frontal cortex while subjects performed two tasks that involve three types of cognitive conflict. Neurons encoding predicted conflict, conflict, and error in one or both tasks were intermixed, forming a representational geometry that simultaneously allowed task specialization and generalization. Neurons encoding conflict retrospectively served to update internal estimates of control demand. Population representations of conflict were compositional. These findings reveal how representations of evaluative signals can be both abstract and task-specific and suggest a neuronal mechanism for estimating control demand.


title: The geometry of domain-general performance monitoring in the human medial frontal cortex


title: FANCD2 directly inhibits DNA2 nuclease at stalled replication forks and acts as a RAD51 mediator in strand exchange

abstract: FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea induced stalled forks. The mechanisms of the fork protection are not well studied. Here, we purified FANCD2 to study how FANCD2 regulates DNA resection at stalled forks. In vitro, we showed that FANCD2 inhibits fork degradation in two ways: 1) it inhibits DNA2 nuclease activity by directly binding to DNA2. 2) independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit various nucleases, including DNA2. More unexpectedly, FANCD2 acts as a RAD51 mediator to stimulate the strand exchange activity of RAD51, and does so by enhancing ssDNA binding of RAD51. Our work biochemically explains mechanisms by which FANCD2 protects stalled forks and further provides a simple molecular explanation for genetic interactions between FANCD2 and the BRCA2 mediator.


title: Soil conditions drive belowground trait space in temperate agricultural grasslands

abstract:
O_LIPlant belowground organs perform essential functions, including water and nutrient uptake, anchorage, vegetative reproduction and recruitment of mutualistic soil microbiota. Determining how belowground traits jointly determine dimensions of the trait space and how these dimensions are linked to environmental conditions would further advance our understanding of plant functioning and community assembly.
C_LIO_LIHere, we investigated belowground plant-trait dimensionality and its variation along 10 soil and land-use parameters in 150 temperate grasslands plots. We used eight belowground traits collected in greenhouse and common garden experiments, as well as bud-bank size and specific leaf area from databases, for a total of 313 species, to calculate community weighted means (CWMs).
C_LIO_LIUsing PCA, we found that about 55% of variance in CWMs was explained by two main dimensions, corresponding to a mycorrhizal  collaboration and a resource  conservation gradient. Frequently overlooked traits such as rooting depth, bud-bank size and root branching intensity were largely integrated in this bidimensional trait space. The two plant-strategy gradients were partially dependent on each other, with  outsourcing communities along the collaboration gradient being more often  slow. These  outsourcing communities were also more often deep-rooting, and associated with soil parameters, such as low moisture and sand content, high topsoil pH, high C:N and low {delta}15N.  Slow communities had large bud-banks and were associated with low land-use intensity, high topsoil pH, and low nitrate but high ammonium concentrations in the soil. We did not find a substantial role of phosphorus-availability as an indicator along the  collaboration gradient.
C_LIO_LIIn conclusion, the  collaboration and  conservation gradients previously identified at the species level scale up to community level in grasslands, encompass more traits than previously described, and vary with the environment.
C_LI

category: ecology


title: Gene co-expression reveals the modularity and integration of C4 and CAM in Portulaca

abstract: C4 photosynthesis and Crassulacean acid metabolism (CAM) have been considered as largely independent adaptations in spite of sharing key biochemical modules. Portulaca is a geographically widespread clade of over 100 annual and perennial angiosperm species that primarily use C4, but facultatively exhibit CAM when drought stressed, a photosynthetic system known as C4+CAM. It has been hypothesized that C4+CAM is rare because of pleiotropic constraints, but these have not been deeply explored. We generated a chromosome-level genome assembly of P. amilis and sampled mRNA from P. amilis and P. oleracea during CAM induction. Gene co-expression network analyses identified C4 and CAM gene modules shared and unique to both Portulaca species. A conserved CAM module linked phosphoenolpyruvate carboxylase (PEPC) to starch turnover during the day-night transition and was enriched in circadian clock regulatory motifs in the P. amilis genome. Preservation of this co-expression module regardless of water status suggests that Portulaca constitutively operate a weak CAM cycle that is transcriptionally and post-transcriptionally upregulated during drought. C4 and CAM mostly used mutually exclusive genes for primary carbon fixation and it is likely that nocturnal CAM malate stores are shuttled into diurnal C4 decarboxylation pathways, but we find evidence that metabolite cycling may occur at low levels. C4 likely evolved in Portulaca through co-option of redundant genes and integration of the diurnal portion of CAM. Thus, the ancestral CAM system did not strongly constrain C4 evolution because photosynthetic gene networks are not co-regulated for both daytime and nighttime functions.


title: Causes of variability in estimates of mutational variance from mutation accumulation experiments

abstract: Characteristics of the new phenotypic variation introduced via mutation have broad implications in evolutionary and medical genetics. Standardised estimates of this mutational variance, VM, span two orders of magnitude, but the causes of this remain poorly resolved. We investigated estimate heterogeneity using two approaches. First, meta-analyses of ~150 estimates from 37 mutation accumulation (MA) studies did not support a difference among taxa (which differ in mutation rate) in standardised VM, but provided equivocal support for standardised VM to vary with trait type (life history versus morphology, predicted to differ in mutation rate). Notably, several experimental factors were confounded with taxon and trait, and further empirical data are required to resolve their influences. Second, we analysed morphological data from an experiment in Drosophila serrata to determine the potential for unintentional heterogeneity among environments in which phenotypes were measured (i.e., among laboratories or time points) or transient segregation of mutations within MA lines to affect standardised VM. Approximating the size of an average MA experiment, variability among repeated estimates of (accumulated) mutational variance was comparable to variation among published estimates of standardised VM for morphological traits. This heterogeneity was (partially) attributable to unintended environmental variation or within line segregation of mutations only for wing size, not wing shape traits. We conclude that sampling error contributed substantial variation within this experiment, and infer that it will also contribute substantially to differences among published estimates. We suggest a logistically permissive approach to improve the precision of estimates, and consequently our understanding of the dynamics of mutational variance of quantitative traits.

category: genetics


title: The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

abstract:
The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+ and Ca2+, and thus shapes cellular excitability, plasticity and metabolic activity. The molecular appearance and operation of TRPM7 channel complexes in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative MS analysis. We found that native TRPM7 channels are high molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ARL15. Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that ARL15 effectively and specifically impacts TRPM7 channel activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

Impact StatementHigh-resolution proteomics in conjunction with biochemical and electrophysiological experiments revealed that the channel-kinase TRPM7 in the rodent brain forms macromolecular complexes containing the metal transporters CNNM1-4 and a small G protein ARL15.


title: Dug: A Semantic Search Engine Leveraging Peer-Reviewed Literature to Span Biomedical Data Repositories

abstract:
MotivationAs the number of public data resources continues to proliferate, identifying relevant datasets across heterogenous repositories is becoming critical to answering scientific questions. To help researchers navigate this data landscape, we developed Dug: a semantic search tool for biomedical datasets utilizing evidence-based relationships from curated knowledge graphs to find relevant datasets and explain why those results are returned.

ResultsDeveloped through the National Heart, Lung, and Blood Institutes (NHLBI) BioData Catalyst ecosystem, Dug has indexed more than 15,911 study variables from public datasets. On a manually curated search dataset, Dugs total recall (total relevant results/total results) of 0.79 outperformed default Elasticsearchs total recall of 0.76. When using synonyms or related concepts as search queries, Dug (0.36) far outperformed Elasticsearch (0.14) in terms of total recall with no significant loss in the precision of its top results.

Availability and ImplementationDug is freely available at https://github.com/helxplatform/dug. An example Dug deployment is also available for use at https://search.biodatacatalyst.renci.org/.

Contactawaldrop@rti.org or scox@renci.org

category: bioinformatics


title: Dug: A Semantic Search Engine Leveraging Peer-Reviewed Knowledge to Span Biomedical Data Repositories


title: MPA_Pathway_Tool: User-friendly, automatic assignment of microbial community data on metabolic pathways

abstract:
MotivationTaxonomic and functional characterization of microbial communities from diverse environments such as the human gut or biogas plants by multi-omics methods plays an ever more important role. Researchers assign all identified genes, transcripts, or proteins to biological pathways to better understand the function of single species and microbial communities. However, due to the versatility of microbial metabolism and a still increasing number of new biological pathways, linkage to standard pathway maps such as the KEGG (Kyoto Encyclopedia of Genes and Genomes) central carbon metabolism is often problematic.

ResultsWe successfully implemented and validated a new user-friendly, stand-alone web application, the MPA_Pathway_Tool. It consists of two parts, called  Pathway-Creator and  Pathway-Calculator. The  Pathway-Creator enables an easy setup of user-defined pathways with specific taxonomic constraints. The  Pathway-Calculator automatically maps microbial community data from multiple measurements on selected pathways and visualizes the results.

Availability and ImplementationThe MPA_Pathway_Tool is implemented in Java and ReactJS. It is freely available on http://mpa-pathwaymapper.ovgu.de/. Further documentation and the complete source code are available on GitHub (https://github.com/danielwalke/MPA_Pathway_Tool).

Contactdaniel.walke@ovgu.de, mailto:heyer@mpi-magdeburg.mpg.de heyer@mpi-magdeburg.mpg.de

Supplementary InformationAdditional files and images are available at MDPI online.

Highlightsuser-friendly generation of pathways, re-using of existent metabolic pathways, automated mapping of data


title: Simulations of dynamically cross-linked actin networks: morphology, rheology, and hydrodynamic interactions

abstract: Cross-linked actin networks are the primary component of the cell cytoskeleton and have been the subject of numerous experimental and modeling studies. While these studies have demonstrated that the networks are viscoelastic materials, evolving from elastic solids on short timescales to viscous fluids on long ones, questions remain about the duration of each asymptotic regime, the role of the surrounding fluid, and the behavior of the networks on intermediate timescales. Here we perform detailed simulations of passively cross-linked non-Brownian actin networks to quantify the principal timescales involved in the elastoviscous behavior, study the role of nonlocal hydrodynamic interactions, and parameterize continuum models from discrete stochastic simulations. To do this, we extend our recent computational framework for semiflexible filament suspensions, which is based on nonlocal slender body theory, to actin networks with dynamic cross linkers and finite filament lifetime. We introduce a model where the cross linkers are elastic springs with sticky ends stochastically binding to and unbinding from the elastic filaments, which randomly turn over at a characteristic rate. We show that, depending on the parameters, the network evolves to a steady state morphology that is either an isotropic actin mesh or a mesh with embedded actin bundles. For different degrees of bundling, we numerically apply small-amplitude oscillatory shear deformation to extract three timescales from networks of hundreds of filaments and cross linkers. We analyze the dependence of these timescales, which range from the order of hundredths of a second to the actin turnover time of several seconds, on the dynamic nature of the links, solvent viscosity, and filament bending stiffness. We show that the network is mostly elastic on the short time scale, with the elasticity coming mainly from the cross links, and viscous on the long time scale, with the effective viscosity originating primarily from stretching and breaking of the cross links. We show that the influence of nonlocal hydrodynamic interactions depends on the network morphology: for homogeneous meshworks, nonlocal hydrodynamics gives only a small correction to the viscous behavior, but for bundled networks it both hinders the formation of bundles and significantly lowers the resistance to shear once bundles are formed. We use our results to construct three-timescale generalized Maxwell models of the networks.


title: Structure and Mechanism of Hedgehog Acyl Transferase

abstract: The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.


title: Structural modeling of the hERG potassium channel and associated drug interactions

abstract:
The voltage-gated potassium channel, KV11.1, encoded by the human Ether-a-go-go- Related Gene (hERG) is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage- dependence of channel inactivation. Because hERG channel blockade is implicated in drug-induced arrhythmias associated with both the open and inactivated states, we used Rosetta to simulate effects of hERG S620T and S641A/T mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and formation of four lateral fenestrations in the pore. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our structural modeling correlate well with existing experimental evidence involving interactions of these drugs with key hERG residues Y652 and F656 inside the pore and reveal potential ligand binding interactions within fenestration region in an inactivated state.

Significance StatementComputational models of hERG potassium channel provide structural insights into an inactivated state and associated drug interactions. Our computational approach will be useful to study ion channel modulation by small molecules.


title: Stress relaxation amplitude of hydrogels determines migration, proliferation, and morphology of cells in 3-D

abstract: The viscoelastic behavior of hydrogel matrices sensitively influences the cell behavior in 3D culture and biofabricated tissue model systems. Previous reports have demonstrated that cells tend to adhere, spread, migrate and proliferate better in hydrogels with pronounced stress relaxation. However, it is currently unknown if cells respond more sensitively to the amplitude of stress relaxation, or to the relaxation time constant. To test this, we compare the behavior of fibroblasts cultured for up to 10 days in alginate and oxidized alginate hydrogels with similar Youngs moduli but diverging stress relaxation behavior. We find that fibroblasts elongate, migrate and proliferate better in hydrogels that display a higher stress relaxation amplitude. By contrast, the cells response to the relaxation time constant was less pronounced and less consistent. Together, these data suggest that it is foremost the stress relaxation amplitude of the matrix that determines the ability of cells to locally penetrate and remodel the matrix, which subsequently leads to better spreading, faster migration, and higher cell proliferation. We conclude that the stress relaxation amplitude is a central design parameter for optimizing cell behavior in 3-D hydrogels.


title: Transcranial photoacoustic characterization of neurovascular physiology during early-stage photothrombotic stroke in neonatal piglets in vivo

abstract: Perinatal ischemic stroke is estimated to occur in 1/2300-1/5000 live births, but early differential diagnosis from global hypoxia-ischemia is often difficult. In this study, we tested the ability of a hand-held transcranial photoacoustic (PA) imaging to non-invasively detect a focal photothrombotic stroke (PTS) within 2 hours of stroke onset in a gyrencephalic piglet brain. 17 stroke lesions of approximately 1-cm2 area were introduced randomly in anterior or posterior cortex via the light/dye PTS technique in anesthetized neonatal piglets (n = 11). The contralateral non-ischemic region served as control tissue for discrimination contrast for the PA hemoglobin metrics: HbO2 saturation, total hemoglobin (tHb), and individual quantities of oxygenated and deoxygenated hemoglobin (HbO2 and HbR). The PA-derived tissue HbO2 saturation at 2 hours yielded a significant separation between control and affected regions-of-interest (p < 0.0001), which were well matched with 24-hr post-stroke cerebral infarction confirmed in the triphenyltetrazolium chloride (TTC)-stained image. The quantity of HbO2 also displayed a significant contrast (p = 0.021), whereas tHb and HbR did not. The analysis on receiver operating characteristic curves and multivariate data analysis also agreed with the results above. This study shows that a hand-held transcranial PA neuroimaging can detect a regional thrombotic stroke in cerebral cortex of a neonatal piglet. In particular, we conclude that the HbO2 saturation metric can be used alone to identify regional stroke lesions. The lack of change in tHb may be related to arbitrary hand-held imaging configuration and/or entrapment of red blood cells within the thrombotic stroke.


title: Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels

abstract: In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+ with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.


title: Protein structural features predict responsiveness to pharmacological chaperone treatment for three lysosomal storage disorders

abstract:
Three-dimensional structures of proteins can provide important clues into the efficacy of personalized treatment. We perform a structural analysis of variants within three inherited lysosomal storage disorders, comparing variants responsive to pharmacological chaperone treatment to those unresponsive to such treatment. We find that predicted {Delta}{Delta}G of mutation is higher on average for variants unresponsive to treatment, in the case of datasets for both Fabry disease and Pompe disease, in line with previous findings. Using both a single decision tree and an advanced machine learning approach based on the larger Fabry dataset, we correctly predict responsiveness of three Gaucher disease variants, and we provide predictions for untested variants. Many variants are predicted to be responsive to treatment, suggesting that drug-based treatments may be effective for a number of variants in Gaucher disease. In our analysis, we observe dependence on a topological feature reporting on contact arrangements which is likely connected to the order of folding of protein residues, and we provide a potential justification for this observation based on steady-state cellular kinetics.

Author summaryPharmacological chaperones are small molecule drugs that bind to proteins to help stabilize the folded state. One set of diseases for which this treatment has been effective is the lysosomal storage disorders, which are caused by defective lysosomal enzymes. However, not all genotypes are equally responsive to treatment. For instance, missense mutants that are particularly destabilized relative to WT are less likely to respond. The availability of datasets containing responsiveness data for large numbers of mutants, along with crystal structures of the protein involved in each disease, make machine learning methods incorporating sequence-based and structural data feasible. We hypothesize that data from two diseases, Fabry and Pompe disease, may be useful for predicting responsiveness of variants in the related Gaucher disease. Results suggest that many rare variants in Gaucher disease could be amenable to existing drugs. Results suggest that drug responsiveness depends on protein topology is such a way that mutations in early-to-fold residues are more likely to be non-responsive to pharmacological chaperone treatment, which is consistent with a simple kinetic model of stability rescue. This study provides an example of how machine learning can be used to inform further studies towards personalized treatment in medicine.