title: Alkaloid and acetogenin-rich fraction from Annona crassiflora fruit peel inhibits proliferation and migration of human liver cancer HepG2 cells

abstract: Plant species from Annonaceae are commonly used in traditional medicine to treat various cancer types. This study aimed to investigate the antiproliferative potential of an alkaloid and acetogenin-rich fraction from the fruit peel of Annona crassiflora in HepG2 cells. A liquid-liquid fractionation was carried out on the ethanol extract of A. crassiflora fruit peel in order to obtain an alkaloid and acetogenin-rich fraction (AF-Ac). Cytotoxicity, proliferation and migration were evaluated in the HepG2 cells, as well as the proliferating cell nuclear antigen (PCNA), vinculin and epidermal growth factor receptor (EGFR) expression. In addition, intracellular Ca2+ was determined using Fluo4-AM and fluorescence microscopy. First, 9 aporphine alkaloids and 4 acetogenins that had not yet been identified in the fruit peel of A. crassiflora were found in AF-Ac. The treatment with 50 {micro}g/mL AF-Ac reduced HepG2 cell viability, proliferation and migration (p < 0.001), which is in accordance with the reduced expression of PCNA and EGFR levels (p < 0.05). Furthermore, AF-Ac increased intracellular Ca2+ in the HepG2 cells, mobilizing intracellular calcium stores, which might be involved in the anti-migration and anti-proliferation capacities of AF-Ac. Our results support the growth-inhibitory potential of AF-Ac on HepG2 cells and suggest that this effect is triggered, at least in part, by PCNA and EGFR modulation and mobilization of intracellular Ca2+. This study showed biological activities not yet described for A. crassiflora fruit peel, which provide new possibilities for further in vivo studies to assess the antitumoral potential of A. crassiflora, especially its fruit peel.


title: Selective abrogation of S6K2 maps lipid homeostasis as a survival vulnerability in MAPKi-resistant NRASmut melanoma

abstract: Although oncogenic NRAS activates MAPK signaling, inhibition of the MAPK pathway is not therapeutically efficacious in NRAS-mutant tumors. Here we report that silencing the ribosomal protein S6 kinase 2 (S6K2), while preserving the activity of S6K1, perturbs lipid metabolism, enhances fatty acid unsaturation, triggers lipid peroxidation and induces cell death selectively in NRAS-mutant melanoma cells that are resistant to MAPK inhibition. S6K2 depletion stimulates SREBP1 activity in an S6K1-dependent manner and relieves suppression of PPAR, triggering apoptosis and ferroptosis. Combining PPAR agonists and polyunsaturated fatty acids phenocopies the effects of S6K2 abrogation, blocking tumor growth. Collectively, our study establishes S6K2 and its effector subnetwork as promising targets for NRAS-mutant melanoma that are resistant to global MAPK pathway inhibitors.

category: cancer biology


title: PFKFB3 DEPLETION ACTIVATES β-CELL REPLICATION BY CELL COMPETITIVE CULLING OF COMPROMISED β-CELLS UNDER STRESS

abstract:
Highly conserved hypoxia-inducible factor 1 alpha (HIF1) and its target 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) play a critical role in the survival of damaged {beta}-cells in type 2 diabetes (T2D) while rendering {beta}-cells non-responsive to glucose stimulation by mitochondrial suppression. HIF1-PFKFB3 is activated in 30-50% of all {beta}-cells in diabetic islets, leaving an open question of whether targeting this pathway may adjust {beta}-cell mass and function to the specific metabolic demands during diabetogenic stress.

Our previous studies of {beta}-cells under amyloidogenic stress by human islet amyloid polypeptide (hIAPP) revealed that PFKFB3 is a metabolic execution arm of the HIF1 pathway with potent implications on Ca2+ homeostasis, metabolome, and mitochondrial form and function.

To discriminate the role of PFKFB3 from HIF1 in vivo, we generated mice with conditional {beta}-cell specific disruption of the Pfkfb3 gene on a hIAPP+/- background and a high-fat diet (HFD) [PFKFB3{beta}KO + diabetogenic stress (DS)].

PFKFB3 disruption in {beta}-cells under diabetogenic stress led to selective purging of hIAPP-damaged {beta}-cells and the disappearance of bihormonal insulin- and glucagon-positive cells, thus compromised {beta}-cells. At the same time, PFKFB3 disruption led to a three-fold increase in {beta}-cell replication resembling control levels as measured with minichromosome maintenance 2 protein (MCM2). PFKFB3 disruption depleted bihormonal cells while increased {beta}-cell replication that was reflected in the increased {beta}-/-cell ratio and maintained {beta}-cell mass. Analysis of metabolic performance indicated comparable glucose intolerance and reduced plasma insulin levels in PFKFB3{beta}KO DS relative to PFKFB3WT DS mice. In the PFKFB3{beta}KO DS group, plasma glucagon levels were reduced compared to PFKFB3WT DS mice and were in line with increased insulin sensitivity. Glucose intolerance in PFKFB3{beta}KO DS mice could be explained by the compensatory expression of HIF1 after disruption of PFKFB3. Our data strongly suggest that the replication and functional recovery of {beta}-cells under diabetogenic stress depend on selective purification of HIF1 and PFKFB3-positive {beta}-cells. Thus, HIF1-PFKFB3-dependent activation of cell competition and purging of compromised {beta}-cells may yield functional competent {beta}-cell mass in diabetes.

category: cell biology


title: Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

abstract: Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.


title: SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney

abstract: COVID-19 (coronavirus disease 2019) caused SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection is a disease affecting several organ systems. A model that captures all clinical symptoms of COVID-19 as well as long-haulers disease is needed. We investigated the host responses associated with infection in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines (IL-6, IL-1beta, and TNF) and type II interferons whereas type I interferons were inhibited. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of mycarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2 (PASC).

category: microbiology


title: Comparable environmental stability and disinfection profiles of the currently circulating SARS-CoV-2 variants of concern B.1.1.7 and B.1.351

abstract: The emergence of novel SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern with increased transmission dynamics has raised questions regarding stability and disinfection of these viruses. In this study, we analyzed surface stability and disinfection of the currently circulating SARS-CoV-2 variants B.1.1.7 and B.1.351 compared to the wildtype. Treatment with heat, soap and ethanol revealed similar inactivation profiles indicative of a comparable susceptibility towards disinfection. Furthermore, we observed comparable surface stability on steel, silver, copper and face masks. Overall, our data support the application of currently recommended hygiene concepts to minimize the risk of B.1.1.7 and B.1.351 transmission.


title: Functional evaluation of proteolytic activation for the SARS-CoV-2 variant B.1.1.7: role of the P681H mutation

abstract:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread.

HighlightsO_LISARS-CoV-2 B.1.1.7 VOC has a P681H mutation in the spike that is predicted to enhance viral infection
C_LIO_LIP681H does not significantly impact furin cleavage, viral entry or cell-cell spread
C_LIO_LIOther mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates
C_LI

Graphical abstract

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title: Functional evaluation of the P681H mutation on the proteolytic activation the SARS-CoV-2 variant B.1.1.7 (Alpha) spike


title: Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape

abstract: An ideal anti-SARS-CoV-2 antibody would resist viral escape1-3, have activity against diverse SARS-related coronaviruses4-7, and be highly protective through viral neutralization8-11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S3094, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

abstract: The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

category: biochemistry


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp12/7/8 RNA-dependent RNA Polymerase

abstract: The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologs in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer (FRET)-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified 3 novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp13 Helicase

abstract: The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterised pharmaceuticals for nsp13 inhibitors using a FRET-based high-throughput screening (HTS) approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

abstract: The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp15 Endoribonuclease

abstract: SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered the economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.


title: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp14/nsp10 Exoribonuclease

abstract: SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.


title: Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

abstract: The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

category: bioengineering


title: Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

abstract: The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

category: synthetic biology


title: TMPRSS2 and RNA-dependent RNA polymerase are effective targets of therapeutic intervention for treatment of COVID-19 caused by SARS-CoV-2 variants (B.1.1.7 and B.1.351)

abstract: SARS-CoV-2 is a causative agent of COVID-19 pandemic and the development of therapeutic interventions is urgently needed. So far, monoclonal antibodies and drug repositioning are the main methods for drug development and this effort was partially successful. Since the beginning of COVID-19 pandemic, the emergence of SARS-CoV-2 variants has been reported in many parts of the world and the main concern is whether the current vaccines and therapeutics are still effective against these variant viruses. The viral entry and viral RNA-dependent RNA polymerase (RdRp) are the main targets of current drug development, thus the inhibitory effects of TMPRSS2 and RdRp inhibitors were compared among the early SARS-CoV-2 isolate (lineage A) and the two recent variants (lineage B.1.1.7 and lineage B.1.351) identified in the UK and South Africa, respectively. Our in vitro analysis of viral replication showed that the drugs targeting TMPRSS2 and RdRp are equally effective against the two variants of concern.


title: Temporal specificity of abnormal neural oscillations during phonatory events in Laryngeal Dystonia

abstract: Laryngeal Dystonia is a debilitating disorder of voicing in which the laryngeal muscles are intermittently in spasm resulting in involuntary interruptions during speech. The central pathophysiology of laryngeal dystonia, underlying computational impairments in vocal motor control, remains poorly understood. Although prior imaging studies have found aberrant activity in the central nervous system during phonation in patients with laryngeal dystonia, it is not known at what timepoints during phonation these abnormalities emerge and what function may be impaired. To investigate this question, we recruited 22 adductor laryngeal dystonia patients (15 female, age range = 28.83-72.46 years) and 18 controls (8 female, age range = 27.40-71.34 years). We leveraged the fine temporal resolution of magnetoencephalography to monitor neural activity around glottal movement onset, subsequent voice onset and after the onset of pitch feedback perturbations. We examined event-related beta-band (12-30 Hz) and high-gamma band (65-150 Hz) neural oscillations. Prior to glottal movement onset, we observed abnormal frontoparietal motor preparatory activity. After glottal movement onset, we observed abnormal activity in somatosensory cortex persisting through voice onset. Prior to voice onset and continuing after, we also observed abnormal activity in the auditory cortex and the cerebellum. After pitch feedback perturbation onset, we observed no differences between controls and patients in their behavioural responses to the perturbation. But in patients, we did find abnormal activity in brain regions thought to be involved in the auditory feedback control of vocal pitch (premotor, motor, somatosensory and auditory cortices). Our study results confirm the abnormal processing of somatosensory feedback that has been seen in other studies. However, there were several remarkable findings in our study. First, patients have impaired vocal motor activity even before glottal movement onset, suggesting abnormal movement preparation. These results are significant because: (i) they occur before movement onset, abnormalities in patients cannot be ascribed to deficits in vocal performance, and (ii) they show that neural abnormalities in laryngeal dystonia are more than just abnormal responses to sensory feedback during phonation as has been hypothesised in some previous studies. Second, abnormal auditory cortical activity in patients begins even before voice onset, suggesting abnormalities in setting up auditory predictions before the arrival of auditory feedback at voice onset. Generally, activation abnormalities identified in key brain regions within the speech motor network around various phonation events not only provide temporal specificity to neuroimaging phenotypes in laryngeal dystonia but also may serve as potential therapeutic targets for neuromodulation.

category: neuroscience


title: Motivational learning biases are differentially modulated by genetic determinants of striatal and prefrontal dopamine function

abstract: Dopaminergic neurotransmission plays a pivotal role in appetitively motivated behavior in mammals, including humans. Notably, action and valence are not independent in motivated tasks, and it is particularly difficult for humans to learn the inhibition of an action to obtain a reward. We have previously observed that the carriers of the DRD2/ANKK1 TaqIA A1 allele, that has been associated with reduced striatal dopamine D2 receptor expression, showed a diminished learning performance when required to learn response inhibition to obtain rewards, a finding that was replicated in two independent cohorts. In the present study, we first report a replication of this finding in a third independent cohort of 99 participants. Interestingly, after combining all three cohorts (total N = 281), exploratory analyses regarding the COMT Val108/158Met polymorphism suggest that homozygotes for the Met allele, which has been linked to higher prefrontal dopaminergic tone, show a lower learning bias. Our results corroborate the importance of genetic variability of the dopaminergic system in individual learning differences of action-valence interaction and, furthermore, suggest that motivational learning biases are differentially modulated by genetic determinants of striatal and prefrontal dopamine function.


title: BDNF Val66Met polymorphism as putative genetic substrate of music-induced plasticity in auditory prediction

abstract: Predictive processing of sounds depends on the constant updating of priors based on exposure to posteriors, which through repeated exposure mediates learning. The result of such corrections to the model is seen in musicians, whose lifelong training results in measurable plasticity of audio-motor brain anatomy and functionality. It has been suggested that the plasticity of auditory predictive processes depends on the interaction between the environment and the individuals genetic substrate. However, empirical evidence to this is still missing. BDNF is a critical genetic factor affecting learning and plasticity, and its widely studied functional variant Val66Met single-nucleotide polymorphism offers a unique opportunity to investigate neuroplastic functional changes occurring upon a years-long training. We hypothesised that BDNF gene variations would be driving neuroplasticity of the auditory cortex in musically trained human participants. To this goal, musicians and non-musicians were recruited and divided in Val/Val and Met carriers and their brain activity measured with magnetoencephalography (MEG) while they listened to a regular auditory sequence containing different types of prediction errors. The auditory cortex responses to prediction errors was enhanced in Val/Val carriers who underwent intensive musical training, compared to Met and non-musicians. Our results point at a role of gene-regulated neurotrophic factors in the neural adaptations of auditory processing after long-term training.


title: Rapid neural encoding of the contrast between native and nonnative speech in the alpha band

abstract:
More than half of the worlds population is multilingual, yet it is not known how the human brain encodes the perception of native vs. nonnative speech. To find out, we asked German native speakers to detect the onset of native and nonnative (English and Turkish) vowels in a roving standard stimulation. Using EEG, we show that nonnativeness is robustly registered by an increase in phase coherence in the alpha band (8-12 Hz), beginning as early as [~]100 ms after stimulus onset and lasting more than 200 ms. The alpha band effect is speech-specific, successfully predicts the response speed advantage of nonnative speech, and grants [~]90% decoding accuracy in distinguishing native vs. nonnative speech irrespective of language familiarity. We propose alpha phase coherence as a candidate neural channel for the online resolution of the native-nonnative contrast in the adult brain.

Significance StatementWe show that the human brain takes only [~] 100 ms to distinguish native vs. nonnative speech. This difference is neurally encoded in the alpha band in a reliable and specific way, supporting a processing model in which the mixture of top-down and bottom-up information represented in the speech signal is concurrently partitioned into different frequency channels.


title: Structural basis for broad sarbecovirus neutralization by a human monoclonal antibody

abstract: The recent emergence of SARS-CoV-2 variants of concern (VOC) and the recurrent spillovers of coronaviruses in the human population highlight the need for broadly neutralizing antibodies that are not affected by the ongoing antigenic drift and that can prevent or treat future zoonotic infections. Here, we describe a human monoclonal antibody (mAb), designated S2x259, recognizing a highly conserved cryptic receptor-binding domain (RBD) epitope and cross-reacting with spikes from all sarbecovirus clades. S2x259 broadly neutralizes spike-mediated entry of SARS-CoV-2 including the B.1.1.7, B.1.351, P.1 and B.1.427/B.1.429 VOC, as well as a wide spectrum of human and zoonotic sarbecoviruses through inhibition of ACE2 binding to the RBD. Furthermore, deep-mutational scanning and in vitro escape selection experiments demonstrate that S2x259 possesses a remarkably high barrier to the emergence of resistance mutants. We show that prophylactic administration of S2x259 protects Syrian hamsters against challenges with the prototypic SARS-CoV-2 and the B.1.351 variant, suggesting this mAb is a promising candidate for the prevention and treatment of emergent VOC and zoonotic infections. Our data unveil a key antigenic site targeted by broadly-neutralizing antibodies and will guide the design of pan-sarbecovirus vaccines.

category: immunology


title: Predicting population genetic change in an experimental stochastic environment

abstract: Most natural environments exhibit a substantial component of random variation. Such environmental noise is expected to cause random fluctuations in natural selection, affecting the predictability of evolution. But despite a long-standing theoretical interest for understanding the population genetic consequences of stochastic environments, there has been a dearth of empirical validation and estimation of the underlying parameters of this theory. Indeed, tracking the genetics of a large number of replicate lines under a controlled level of environmental stochasticity is particularly challenging. Here, we tackled this problem by resorting to an automated experimental evolution approach. We used a liquid-handling robot to expose over a hundred lines of the micro-alga Dunaliella salina to randomly fluctuating salinity over a continuous range, with controlled mean, variance, and autocorrelation. We then tracked the frequency of one of two competing strains through amplicon sequencing of a nuclear and choloroplastic barcode sequences. We show that the magnitude of environmental fluctuations (variance), but also their predictability (autocorrelation), have large impacts on the average selection coefficient. Furthermore, the stochastic variance in population genetic change is substantially higher in a fluctuating environment. Reaction norms of selection coefficients and growth rates of single strains against the environment captured the mean response accurately, but failed to explain the high variance induced by environmental stochasticity. Overall, our results provide exceptional insights on the prospects for understanding and predicting genetic evolution in randomly fluctuating environments.

category: evolutionary biology


title: Effective gene expression prediction from sequence by integrating long-range interactions

abstract:
The next phase of genome biology research requires understanding how DNA sequence encodes phenotypes, from the molecular to organismal levels. How noncoding DNA determines gene expression in different cell types is a major unsolved problem, and critical downstream applications in human genetics depend on improved solutions. Here, we report substantially improved gene expression prediction accuracy from DNA sequence through the use of a new deep learning architecture called Enformer that is able to integrate long-range interactions (up to 100 kb away) in the genome. This improvement yielded more accurate variant effect predictions on gene expression for both natural genetic variants and saturation mutagenesis measured by massively parallel reporter assays. Notably, Enformer outperformed the best team on the critical assessment of genome interpretation (CAGI5) challenge for noncoding variant interpretation with no additional training. Furthermore, Enformer learned to predict promoter-enhancer interactions directly from DNA sequence competitively with methods that take direct experimental data as input. We expect that these advances will enable more effective fine-mapping of growing human disease associations to cell-type-specific gene regulatory mechanisms and provide a framework to interpret cis-regulatory evolution. To foster these downstream applications, we have made the pre-trained Enformer model openly available, and provide pre-computed effect predictions for all common variants in the 1000 Genomes dataset.

One-sentence summaryImproved noncoding variant effect prediction and candidate enhancer prioritization from a more accurate sequence to expression model driven by extended long-range interaction modelling.

category: genomics


title: Transcript- and annotation-guided genome assembly of the European starling

abstract: The European starling, Sturnus vulgaris, is an ecologically significant, globally invasive avian species that is also suffering from a major decline in its native range. Here, we present the genome assembly and long-read transcriptome of an Australian-sourced European starling (S. vulgaris vAU), and a second North American genome (S. vulgaris vNA), as complementary reference genomes for population genetic and evolutionary characterisation. S. vulgaris vAU combined 10x Genomics linked-reads, low-coverage Nanopore sequencing, and PacBio Iso-Seq full-length transcript scaffolding to generate a 1050 Mb assembly on 1,628 scaffolds (72.5 Mb scaffold N50). Species-specific transcript mapping and gene annotation revealed high structural and functional completeness (94.6% BUSCO completeness). Further scaffolding against the high-quality zebra finch (Taeniopygia guttata) genome assigned 98.6% of the assembly to 32 putative nuclear chromosome scaffolds. Rapid, recent advances in sequencing technologies and bioinformatics software have highlighted the need for evidence-based assessment of assembly decisions on a case-by-case basis. Using S. vulgaris vAU, we demonstrate how the multifunctional use of PacBio Iso-Seq transcript data and complementary homology-based annotation of sequential assembly steps (assessed using a new tool, SAAGA) can be used to assess, inform, and validate assembly workflow decisions. We also highlight some counter-intuitive behaviour in traditional BUSCO metrics, and present BO_SCPLOWUSCOMPC_SCPLOW, a complementary tool for assembly comparison designed to be robust to differences in assembly size and base-calling quality. Finally, we present a second starling assembly, S. vulgaris vNA, to facilitate comparative analysis and global genomic research on this ecologically important species.


title: APOE ε4 allele advances the age-dependent decline of amyloid β clearance in the human cortex

abstract:
IntroductionOur previous study indicated that the pericapillary clearance of amyloid {beta} (A{beta}) declines with age in APOE 3/3 subjects. Here, we examine whether the APOE {varepsilon}4 allele has an impact on this age-related decline.

MethodsWe examined 69 autopsy brains of APOE {varepsilon}3/{varepsilon}4 or APOE {varepsilon}3/{varepsilon}3 individuals (30-65 years) for the immunohistochemical localization of intracellular, extracellular, and pericapillary A{beta} in the cerebral cortex.

ResultsIn APOE {varepsilon}3/{varepsilon}4 individuals, the percentage of A{beta} positive pericapillary spaces began to decrease (p=0.030), and the number of extracellular A{beta} particles increased in the early 30s (p=0.0008). Those average values were significantly lower (p<0.0001) and higher (p<0.0001), respectively, compared to APOE {varepsilon}3/{varepsilon}3 individuals.

DiscussionOur observations indicate that APOE {varepsilon}4 allele advances by one decade at the onset of age-related decline in A{beta} glymphatic clearance. This finding supports early clinical intervention and stratification by APOE genotype to prevent A{beta} deposition and AD progression.

category: pathology


title: Activity of CaMKIIa+ dorsal cochlear nucleus neurons are crucial for tinnitus perception but not for tinnitus induction

abstract: The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs and is identified as a potential key structure in the generation of phantom sound perception, especially noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN neurons, Ca2+/Calmodulin kinase 2 (CaMKII) positive DCN neurons, using Gi-coupled human M4 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their role in noise-induced tinnitus. Mice were exposed to loud noise (9-11kHz, 90dBSPL, 1h, followed by 2h of silence) and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) were recorded two days before and two weeks after noise exposure to identify animals with a significantly decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of CaMKII+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide (CNO). We found that acutely decreasing firing rate of CaMKII+ DCN units decrease tinnitus-like responses (p = 0.038, n = 11 mice), compared to the control group that showed no improvement in GPIAS (control virus; CaMKII-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to decrease unit firing frequency of CaMKII-hM4Di+ mice and alter best frequency and tuning width of response to sound. However, these effects were not seen if CNO had been previously administered during the noise exposure (n = 6 experimental and 6 control mice). Our results suggest that CaMKII-hM4Di positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining tinnitus perception in mice.


title: Chemogenetically decreasing activity of the dorsal cochlear nucleus can ameliorate noise-induced tinnitus in mice


title: The meta-gut: Hippo inputs lead to community coalescence of animal and environmental microbiomes.

abstract:
All animals carry specialized microbiomes, and their gut microbiotas in particular are continuously released into the environment through excretion of waste. Here we propose the meta-gut as a novel conceptual framework that addresses the ability of the gut microbiome released from an animal to function outside the host and potentially alter ecosystem processes mediated by microbes. An example considered here is the hippopotamus (hippo) and the pools they inhabit. Hippo pool biogeochemistry and fecal and pool water microbial communities were examined through field sampling and an experiment. Sequencing using 16S RNA methods revealed that the active microbial communities in hippo pools that received high inputs of hippo feces are more similar to the hippo gut microbiome than other nearby aquatic environments. The overlap between the microbiomes of the hippo gut and the waters into which they excrete therefore constitutes a meta-gut system with potentially strong influence on the biogeochemistry of pools and downstream waters. We propose that the meta-gut may be present where other species congregate in high densities, particularly in aquatic environments.

SignificanceAnimals can have considerable impacts on biogeochemical cycles and ecosystem attributes through the consumption of resources and physical modifications of the environment. Likewise, microbial communities are well known to regulate biogeochemical cycles. This study links those two observations by showing that the gut microbiome in waste excreted by hippos can persist ex-situ in the environment and potentially alter biogeochemical cycles. This "meta-gut" system may be present in other ecosystems where animals congregate, and may have been more widespread in the past before many large animal populations were reduced in range and abundance.

category: ecology


title: Caspar, an adapter for VAP and TER94, delays the progression of disease by regulating glial inflammation in a Drosophila  model of ALS8

abstract:
Amyotrophic Lateral Sclerosis (ALS) is a fatal, late onset, progressive motor neurodegenerative disorder. We have been studying cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model, which mimics many systemic aspects of the human disease. Here, we show that the ER resident VAPB interacts with Caspar, an ortholog of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly ortholog of ALS14 (VCP/p97, Valosin-containing protein), via its UBX domain and poly-ubiqutinated proteins with its UBA domain. Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 model, a phenomenon that we ascribe to its ability to restrain age-dependant inflammation, modulated by Relish/NF{kappa}B signalling.

We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. Caspar connects the plasma membrane (PM) localized immune signalosome to the ER based VAPB degradative machinery, presumably at PM:ER contact sites. The Caspar:TER94:VAPB complex appears to be a strong candidate for regulating both protein homeostasis and NF{kappa}B signalling. These, in turn, regulate glial inflammation and determine progression of disease. Our study projects human FAF1 as an important protein target to alleviate the progression of motor neuron disease.

category: genetics


title: Discovery of target genes and pathways of blood trait loci using pooled CRISPR screens and single cell RNA sequencing

abstract: The majority of variants associated with complex traits and common diseases identified by genome-wide association studies (GWAS) map to noncoding regions of the genome with unknown regulatory effects in cis and trans. By leveraging biobank-scale GWAS data, massively parallel CRISPR screens and single cell transcriptome sequencing, we discovered target genes of noncoding variants for blood trait loci. The closest gene was often the target gene, but this was not always the case. We also identified trans-effects networks of noncoding variants when cis target genes encoded transcription factors, such as GFI1B and NFE2. We observed that GFI1B trans-target genes were enriched for GFI1B binding sites and fine-mapped GWAS variants, and expressed in human bone marrow progenitor cells, suggesting that GFI1B acts as a master regulator of blood traits. This platform will enable massively parallel assays to catalog the target genes of human noncoding variants in both cis and trans.


title: Tracing patterns of evolution and acquisition of drug resistant Aspergillus fumigatus infection from the environment using population genomics

abstract: Infections caused by opportunistic fungal pathogens are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about the extent to which susceptible patients acquire infection from drug resistant genotypes in the environment. Here, we present a population genomic analysis of the mould Aspergillus fumigatus from across the United Kingdom and Republic of Ireland. First, we show occurrences where azole resistant isolates of near identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Second, we find that the fungus is structured into two clades ( A and  B) with little interclade recombination and the majority of environmental azole resistance genetically clustered inside Clade A. Genome-scans show the impact of selective sweeps across multiple regions of the genome. These signatures of positive selection are seen in regions containing canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, whilst other regions under selection have no defined function. Phenotyping identified genes in these regions that could act as modifiers of resistance showing the utility of reverse genetic approaches to dissect the complex genomic architecture of fungal drug resistance. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.


title: Prior Pro-inflammatory Polarization Changes the Macrophage Response to IL-4

abstract:
Tissue repair is largely regulated by diverse macrophage populations whose functions are timing- and context-dependent. The early phase of healing is dominated by pro-inflammatory macrophages, also known as M1, followed by the emergence of a distinct and diverse population that is collectively referred to as M2. The extent of the diversity of the M2 population is unknown. M2 macrophages may originate directly from circulating monocytes or from phenotypic switching of pre-existing M1 macrophages within the site of injury. The differences between these groups have not been investigated, but have major implications for understanding and treating pathologies characterized by deficient M2 activation, such as chronic wounds, which also exhibit diminished M1 macrophage behavior. This study investigated the influence of prior M1 activation on human macrophage polarization to an M2 phenotype in response to IL-4 treatment in vitro. Compared to unactivated (M0) macrophages, M1 macrophages upregulated several receptors that promote the M2 phenotype, including the primary receptor for IL-4. M1 activation also changed the macrophage response to treatment with IL-4, generating an M2-like phenotype with a distinct gene and protein expression signature compared to M2 macrophages prepared directly from M0 macrophages. Functionally, compared to M0-derived M2 macrophages, M1-derived M2 macrophages demonstrated increased migratory response to SDF-1, and conditioned media from these macrophages promoted increased recruitment of endothelial cells in transwell assays. Together, these findings indicate the importance of prior M1 activation in regulating subsequent M2 behavior, and suggest that augmentation of M1 behavior may be a therapeutic target in dysfunctional tissue repair.

Summary sentenceM1 macrophages that are switched to an M2 phenotype exhibit a distinct functional phenotype compared to M2 macrophages derived directly from unactivated (M0) macrophages.


title: Transcriptional network of the industrial hybrid Saccharomyces pastorianus reveals temperature-dependent allele expression bias and preferential orthologous protein assemblies

abstract: Saccharomyces pastorianus is an industrial natural yeast evolved from different hybridisation events between the mesophilic S. cerevisiae and the cold-tolerant S. eubayanus. This complex aneuploid hybrid carries multiple copies of the parental alleles alongside specific hybrid genes and encodes for multiple protein isoforms which impart novel phenotypes, such as the strong ability to ferment at low temperature. These characteristics lead to agonistic or antagonistic competition for substrates and a plethora of biochemical activities, resulting in a unique cellular metabolism. Here, we investigated the transcriptional signature of the different orthologous alleles in S. pastorianus during temperature shifts. We identified temperature-dependent media-independent genes and showed that 35% have their regulation dependent on extracellular leucine uptake, suggesting an interplay between leucine metabolism and temperature response. The analysis of the expression of ortholog parental alleles unveiled that the majority of the genes express preferentially one parental allele over the other, and that S. eubayanus-like alleles are significantly over-represented among the genes involved in cold acclimatisation. The presence of functionally redundant parental alleles may impact on the nature of protein complexes established in the hybrid, where both parental alleles are competing. Our expression data indicate that the majority of the protein complexes in the hybrid are likely to be either exclusively chimeric or uni-specific, and that the redundancy is discouraged, a scenario which fits well with the stoichiometric balance-hypothesis. This study offers the first overview of the transcriptional pattern of S. pastorianus and provide a rationalisation for its unique industrial traits at expression level.


title: Comparative analysis of Pure Hubs and Pure Bottlenecks in Human Protein-protein Interaction Networks

abstract: Bottlenecks and hubs form a set of topologically important nodes in a network. In this communication, we have made a detailed investigation on hubs and bottlenecks in human protein-protein interaction networks. We find that, three distinct groups exist which we refer to as: a) pure hubs (PHs, nodes having high degree but low betweenness values), b) mix proteins (MXs, nodes having both high degree and high betweenness values) and c) pure bottlenecks (PBs, nodes having high betweenness values but low degree values). Our investigations have revealed that pure hubs, as compared with MXs and PBs, (i) are more disordered, (ii) have higher potential to bind to multiple partners, (iii) are enriched with essential proteins as well as enriched with a higher number of splice variants. The MX proteins, as compared with PHs and PBs, (i) show slower evolutionary patterns, (ii) are involved in multiple pathways, (iii) enriched with the products of genes associated with various diseases and (iv) are more often targeted by bacteria, viruses, protozoa, and fungi pathogens. PBs, as compared with the PHs and MXs, (i) are associated with cancer genes and (ii) are the targets or the nearest neighbors of the targets of most of the approved drugs. Furthermore, our study revealed that these three categories of proteins are involved in distinct functional roles; PHs are involved in housekeeping processes such as transcription and replication; MXs proteins are involved in core signaling pathways whereas PBs are involved in signal transduction processes. Our work, therefore, has identified the distinct characteristics features associated with pure hubs, mix proteins and pure bottlenecks and thus helps in prioritizing proteins based on their degree and betweenness centrality values.

category: systems biology


title: White Matter Hyperintensities, Grey Matter Atrophy, and Cognitive Decline in Neurodegenerative Diseases

abstract:
IntroductionWhite matter hyperintensities (WMHs) as seen on T2w and FLAIR scans represent small-vessel disease related changes in the brain. WMHs are associated with cognitive decline in the normal aging population in general and more specifically in patients with neurodegenerative diseases. In this study, we assessed the different spatial patterns and relationships between WMHs and grey matter (GM) atrophy in normal aging, individuals with mild cognitive impairment (MCI), Alzheimers dementia (AD), fronto-temporal dementia (FTD), and de novo Parkinsons disease (PD).

MethodsImaging and clinical data were obtained from 3 large multi-center databases: The Alzheimers Disease Neuroimaging Initiative (ADNI), the frontotemporal lobar degeneration neuroimaging initiative (NIFD), and the Parkinsons Progression Markers Initiative (PPMI). WMHs and GM atrophy maps were measured in normal controls (N= 571), MCI (N= 577), AD (N= 222), FTD (N= 144), and PD (N= 363). WMHs were segmented using T1w and T2w/PD or FLAIR images and mapped onto 45 white matter tracts using the Yeh WM atlas. GM volume was estimated from the Jacobian determinant of the nonlinear deformation field required to map the subjects MRI to a standard template. The CerebrA atlas was used to obtain volume estimates in 84 GM regions. Mixed effects models were used to compare WMH in different WM tracts and volume of multiple GM structures between patients and controls, assess the relationship between regional WMHs and GM loss for each disease, and investigate their impact on cognition.

ResultsMCI, AD, and FTD patients had significantly higher WMH loads than the matched controls. There was no significant difference in WMHs between PD and controls. For each cohort, significant interactions between WMH load and GM atrophy were found for several regions and tracts, reflecting additional contribution of WMH burden to GM atrophy. While these associations were more relevant for insular and parieto-occipital regions in MCI and AD cohorts, WMH burden in FTD subjects had greater impact on frontal and basal ganglia atrophy. Finally, we found additional contribution of WMH burden to cognitive deficits in AD and FTD subjects compared with matched controls, whereas their impact on cognitive performance in MCI and PD were not significantly different from controls.

ConclusionsWMHs occur more extensively in MCI, AD, and FTD patients than age-matched normal controls. WMH burden on WM tracts also correlates with regional GM atrophy in regions anatomically and functionally related to those tracts, suggesting a potential involvement of WMHs in the neurodegenerative process.


title: Non-invasive real-time access to the output of the spinal cord via a wrist wearable interface

abstract: Despite the promising features of neural interfaces, their trade-off between information transfer and invasiveness has limited translation and viability outside research settings. Here, we present a non-invasive neural interface that provides access to spinal motoneuron activities from a sensor band at the wrist. The interface decodes electric signals present at the tendon endings of the forearm muscles by using a model of signal generation and deconvolution. First, we evaluated the reliability of the interface to detect motoneuron firings, and thereafter we used the decoded neural activity for the prediction of finger movements in offline and real-time conditions. The results showed that motoneuron activity decoded from the wrist accurately predicted individual and combined finger commands and therefore allowed for highly accurate real-time control. These findings demonstrate the feasibility of a wearable, non-invasive, neural interface at the wrist for precise real-time control based on the output of the spinal cord.


title: Juvenile emperor penguin range calls for extended conservation measures in the Southern Ocean

abstract: To protect the unique Southern Ocean biodiversity, conservation measures like marine protected areas (MPAs) are implemented based on the known habitat distribution of ecologically important species. However, distribution models focus on adults, neglecting that immatures animals can inhabit vastly different areas. Here, we show that current conservation efforts in the Southern Ocean are insufficient for ensuring the protection of the highly mobile Emperor penguin. We find that juveniles spend [~]90% of their time outside the boundaries of proposed and existing MPAs, and that their distribution extends far beyond (> 1500 km) the species extent of occurrence as defined by the International Union for Conservation of Nature. We argue that strategic conservation plans for Emperor penguin and long-lived ecologically important species must consider the dynamic habitat range of all age classes.


title: Stable motifs delay species loss in simulated food webs

abstract: Some three-species motifs (unique patterns of interactions between three species) are both more stable when modeled in isolation and over-represented in empirical food webs. This suggests that these motifs may reduce extinction risk for species participating in them, ultimately stabilizing the food web as a whole. We test whether a species time to extinction following a perturbation is related to its participation in stable and unstable motifs and assess how motif roles co-vary with a species degree or trophic level. We found that species motif roles are related to their times to extinction following a disturbance. Specifically, participating in many omnivory motifs (whether in absolute terms, as a proportion of the species role, or relative to other species in the network) was associated with more rapid extinction, even though omnivory has previously been identified as a stable motif. Participating in the other three stable motifs (three-species chain, apparent competition, and direct competition) was associated with longer times to extinction. While motif roles were associated with extinction risk, they also varied strongly with degree and trophic level. This means that these simpler measures of a species role may be sufficient to roughly predict which species are most vulnerable to disturbance, but the additional information encapsulated in a motif role can further refine predictions of vulnerability. Moreover, where researchers are a priori interested in motif roles, our results confirm that these roles can be interpreted with respect to extinction risk.


title: Fungal communities from geothermal soils in Yellowstone National Park

abstract: Geothermal soils offer unique insight into the way extreme environmental factors shape communities of organisms. However, little is known about the fungi growing in these environments and in particular how localized steep abiotic gradients affect fungal diversity. We used metabarcoding to characterize soil fungi surrounding a hot spring-fed thermal creek with water up to 84 {degrees}C and pH 10 in Yellowstone National Park. We found a significant association between fungal communities and soil variable principal components, and we identify the key trends in co-varying soil variables that explain the variation in fungal community. Saprotrophic and ectomycorrhizal fungi community profiles followed, and were significantly associated with, different soil variable principal components, highlighting potential differences in the factors that structure these different fungal trophic guilds. In addition, in vitro growth experiments in four target fungal species revealed a wide range of tolerances to pH levels but not to heat. Overall, our results documenting turnover in fungal species within a few hundred meters suggest many co-varying environmental factors structure the diverse fungal communities found in the soils of Yellowstone National Park.


title: Fungal community turnover along steep environmental gradients from geothermal soils in Yellowstone National Park


title: Highly parallel genomic selection response in replicated Drosophila melanogaster  populations with reduced genetic variation

abstract: Many adaptive traits are polygenic and frequently more loci contributing to the phenotype than needed are segregating in populations to express a phenotypic optimum. Experimental evolution provides a powerful approach to study polygenic adaptation using replicated populations adapting to a new controlled environment. Since genetic redundancy often results in non-parallel selection responses among replicates, we propose a modified Evolve and Resequencing (E&R) design that maximizes the similarity among replicates. Rather than starting from many founders, we only use two inbred Drosophila melanogaster strains and expose them to a very extreme, hot temperature environment (29{degrees}C). After 20 generations, we detect many genomic regions with a strong, highly parallel selection response in 10 evolved replicates. The X chromosome has a more pronounced selection response than the autosomes, which may be attributed to dominance effects. Furthermore, we find that the median selection coefficient for all chromosomes is higher in our two-genotype experiment than in classic E&R studies. Since two random genomes harbor sufficient variation for adaptive responses, we propose that this approach is particularly well-suited for the analysis of polygenic adaptation.


title: Bonsai: An efficient method for inferring large human pedigrees from genotype data

abstract: 1.AO_SCPLOWBSTRACTC_SCPLOWPedigree inference from genotype data is a challenging problem, particularly when pedigrees are sparsely sampled and individuals may be distantly related to their closest genotyped relatives. We present a new method that infers small pedigrees of close relatives and then assembles them into larger pedigrees. To assemble large pedigrees, we introduce several new formulas and tools including a new likelihood for the degree separating two small pedigrees, a method for detecting individuals who share background identity-by-descent (IBD) that does not reflect recent common ancestry, and a method for identifying the ancestral branches through which distant relatives are connected. Our method also takes several new approaches that help to improve the accuracy and efficiency of pedigree inference. In particular, we incorporate age information directly into the likelihood rather than using ages only for consistency checks and we employ a heuristic branch-and-bound-like approach to more efficiently explore the space of possible pedigrees. Together, these approaches make it possible to construct large pedigrees that are challenging or intractable for current inference methods. The new method, Bonsai, is available at https://github.com/23andMe/bonsaitree.


title: Parametric Deconvolution for Cancer Cells Viscoelasticity Measurements from Quantitative Phase Images

abstract: In this contribution, we focused on optimising a dynamic flow-based shear stress system to achieve a reliable platform for cell shear modulus (stiffness) and viscosity assessment using quantitative phase imaging. The estimation of cell viscoelastic properties is influenced by distortion of the shear stress waveform, which is caused by the properties of the flow system components (i.e., syringe, flow chamber and tubing). We observed that these components have a significant influence on the measured cell viscoelastic characteristics. To suppress this effect, we applied a correction method utilizing parametric deconvolution of the flow systems optimized impulse response. Achieved results were compared with the direct fitting of the Kelvin-Voigt viscoelastic model and the basic steady-state model. The results showed that our novel parametric deconvolution approach is more robust and provides a more reliable estimation of viscosity with respect to changes in the syringes compliance compared to Kelvin-Voigt model.


title: Inflammatory and regenerative processes in bioresorbable synthetic pulmonary valves up to 2 years in sheep: Spatiotemporal insights augmented by Raman microspectroscopy

abstract: In situ heart valve tissue engineering is an emerging approach in which resorbable, off-the-shelf available scaffolds are used to induce endogenous heart valve restoration. Such scaffolds are designed to recruit endogenous cells in vivo, which subsequently resorb polymer and produce and remodel new valvular tissue in situ. Recently, preclinical studies using electrospun supramolecular elastomeric valvular grafts have shown that this approach enables in situ regeneration of pulmonary valves with long-term functionality in vivo. However, the evolution and mechanisms of inflammation, polymer absorption and tissue regeneration are largely unknown, and adverse valve remodeling and intra- and inter-valvular variability have been reported. Therefore, the goal of the present study was to gain a mechanistic understanding of the in vivo regenerative processes by combining routine histology and immunohistochemistry, using a comprehensive sheep-specific antibody panel, with Raman microspectroscopy for the spatiotemporal analysis of in situ tissue-engineered pulmonary valves with follow-up to 24 months from a previous preclinical study in sheep. The analyses revealed a strong spatial heterogeneity in the influx of inflammatory cells, graft resorption, and foreign body giant cells. Collagen maturation occurred predominantly between 6 and 12 months after implantation, which was accompanied by a progressive switch to a more quiescent phenotype of infiltrating cells with properties of valvular interstitial cells. Variability among specimens in the extent of tissue remodeling was observed for follow-up times after 6 months. Taken together, these findings advance the understanding of key events and mechanisms in material-driven in situ heart valve tissue engineering.


title: Construction of continuously expandable single-cell atlases through integration of heterogeneous datasets in a generalized cell-embedding space

abstract: Computational tools for integrative analyses of diverse single-cell experiments are facing formidable new challenges including dramatic increases in data scale, sample heterogeneity, and the need to informatively cross-reference new data with foundational datasets. Here, we present SCALEX, a deep-learning method that integrates single-cell data by projecting cells into a batch-invariant, common cell-embedding space in a truly online manner (i.e., without retraining the model). SCALEX substantially outperforms online iNMF and other state-of-the-art non-online integration methods on benchmark single-cell datasets of diverse modalities, (e.g., scRNA-seq, scATAC-seq), especially for datasets with partial overlaps, accurately aligning similar cell populations while retaining true biological differences. We showcase SCALEXs advantages by constructing continuously expandable single-cell atlases for human, mouse, and COVID-19 patients, each assembled from diverse data sources and growing with every new data. The online data integration capacity and superior performance makes SCALEX particularly appropriate for large-scale single-cell applications to build-upon previously hard-won scientific insights.

category: bioinformatics


title: Online single-cell data integration through projecting heterogeneous datasets into a common cell-embedding space


title: plantR: An R package and workflow for managingspecies records from biological collections

abstract:
O_LISpecies records from biological collections are becoming increasingly available online. This unprecedented availability of records has largely supported recent studies in taxonomy, biogeography, macroecology, and biodiversity conservation. Biological collections vary in their documentation and notation standards, which have changed through time. For different reasons, neither collections nor data repositories perform the editing, formatting, and standardization of the data, leaving these tasks to the final users of the species records (e.g. taxonomists, ecologists and conservationists). These tasks are challenging, particularly when working with millions of records from hundreds of biological collections.
C_LIO_LITo help collection curators and final users perform those tasks, we introduce plantR, an open-source package that provides a comprehensive tool-box to manage species records from biological collections. The package is accompanied by the proposal of a reproducible workflow to manage this type of data in taxonomy, ecology, and biodiversity conservation. It is implemented in R and designed to handle relatively large data sets as fast as possible. Initially designed to handle plant species records, many of the plantR features also apply to other groups of organisms, given that the data structure is similar.
C_LIO_LIThe plantR workflow includes tools to (1) download records from different data repositories, (2) standardize typical fields associated with species records, (3) validate the locality, geographical coordinates, taxonomic nomenclature, and species identifications, including the retrieval of duplicates across collections, and (4) summarize and export records, including the construction of species checklists with vouchers.
C_LIO_LIOther R packages provide tools to tackle some of the workflow steps described above. But in addition to the new features and resources related to the data editing and validation, the greatest strength of plantR is to provide a comprehensive and user-friendly workflow in one single environment, performing all tasks from data retrieval to export. Thus, plantR can help researchers better assess data quality and avoid data leakage in a wide variety of studies using species records.
C_LI


title: plantR: An R package and workflow for managing species records from biological collections


title: A rarefaction-without-resampling extension ofPERMANOVA for testing presence-absence associations in the microbiome

abstract:
BackgroundPERMANOVA [1] is currently the most commonly used method for testing community-level hypotheses about microbiome associations with covariates of interest. PERMANOVA can test for associations that result from changes in which taxa are present or absent by using the Jaccard or unweighted UniFrac distance. However, such presence-absence analyses face a unique challenge: confounding by library size (total sample read count), which occurs when library size is associated with covariates in the analysis. It is known that rarefaction (subsampling to a common library size) controls this bias, but at the potential costs of information loss and the introduction of a stochastic component into the analysis.

MethodsHere we develop a non-stochastic approach to PERMANOVA presence-absence analyses that aggregates information over all potential rarefaction replicates without actual resampling, when the Jaccard or unweighted UniFrac distance is used. We compare this new approach to three possible ways of aggregating PERMANOVA over multiple rarefactions obtained from resampling: averaging the distance matrix, averaging the (element-wise) squared distance matrix, and averaging the F-statistic.

ResultsOur simulations indicate that our non-stochastic approach is robust to confounding by library size and outperforms each of the stochastic resampling approaches. We also show that, when overdispersion is low, averaging the (element-wise) squared distance outperforms averaging the unsquared distance, currently implemented in the R package vegan. We illustrate our methods using an analysis of data on inflammatory bowel disease (IBD) in which samples from case participants have systematically smaller library sizes than samples from control participants.


title: Multi-scale predictive modeling discovers Ibudilast as a polypharmacological agent to improve hippocampal dependent spatial learning and memory and mitigate plaque and tangle pathology in a transgenic rat model of Alzheimer's disease

abstract: Alzheimers disease (AD) is a multifactorial disease that exhibits cognitive deficits, neuronal loss, amyloid plaques, neurofibrillary tangles and neuroinflammation in the brain. We developed a multi-scale predictive modeling strategy that integrates machine learning with biophysics and systems pharmacology to model drug actions from molecular interactions to phenotypic responses. We predicted that ibudilast (IBU), a phosphodiesterase inhibitor and toll-like receptor 4 (TLR4) antagonist, inhibited multiple kinases (e.g., IRAK1 and GSG2) as off-targets, modulated multiple AD-associated pathways, and reversed AD molecular phenotypes. We address for the first time the efficacy of ibudilast (IBU) in a transgenic rat model of AD. IBU-treated transgenic rats showed improved cognition and reduced hallmarks of AD pathology. RNA sequencing analyses in the hippocampus showed that IBU affected the expression of pro-inflammatory genes in the TLR signaling pathway. Our results identify IBU as a potential therapeutic to be repurposed for reducing neuroinflammation in AD by targeting TLR signaling.

category: pharmacology and toxicology


title: The role of petal transpiration in floral humidity generation

abstract:
Floral humidity, an area of elevated humidity in the headspace of flowers, has been detected across angiosperms and may function as a pollinator cue for insect pollinators. It is believed floral humidity is produced predominantly through a combination of evaporation of both liquid nectar and transpirational water loss from the flower. However, the role of transpiration in floral humidity generation has not been tested and is largely inferred by continued humidity production when nectar is removed from flowers. Understanding the extent that transpiration contributes to floral humidity has important implications for understanding the function of floral humidity. We test whether transpiration contributes to the floral humidity generation of two species previously identified to produce elevated floral humidity, Calystegia silvatica and Eschscholzia californica. Floral humidity production of flowers that underwent an antitranspirant treatment, petrolatum gel which blocks transpiration from treated tissues, is compared to flowers that did not receive such treatments. Gel treatments reduced floral humidity production to approximately a third of that produced by untreated flowers in C. silvatica, and half of that in E. californica. This confirms, the previously untested, inferences that transpiration has a large contribution to floral humidity generation and that this contribution may vary between species.

HIGHLIGHTWe confirm, the previously untested, inferences that transpiration has a large contribution to floral humidity generation and show that this contribution may vary between species.

category: plant biology


title: Pre-Stimulus Alpha-Band Phase Gates Afferent Visual Cortex Responses

abstract: The neurogenesis of alpha-band (8-13 Hz) activity has been characterized across many different animal experiments. However, the functional role that alpha oscillations play in perception and behavior has largely been attributed to two contrasting hypotheses, with human evidence in favor of either (or both or neither) remaining sparse. On the one hand, alpha generators have been observed in relay sectors of the visual thalamus and are postulated to phasically inhibit afferent visual input in a feedforward manner 1-4. On the other hand, evidence also suggests that the direction of influence of alpha activity propagates backwards along the visual hierarchy, reflecting a feedback influence upon the visual cortex 5-9. The primary source of human evidence regarding the role of alpha phase in visual processing has been on perceptual reports 10-16, which could be modulated either by feedforward or feedback alpha activity. Thus, although these two hypotheses are not mutually exclusive, human evidence clearly supporting either one is lacking. Here, we present human subjects with large, high-contrast visual stimuli that elicit robust C1 event-related potentials (ERP), which peak between 70-80 milliseconds post-stimulus and are thought to reflect afferent primary visual cortex (V1) input17-20. We find that the phase of ongoing alpha oscillations modulates the global field power (GFP) of the EEG during this first volley of stimulus processing (the C1 time-window). On the standard assumption21-23 that this early activity reflects postsynaptic potentials being relayed to visual cortex from the thalamus, our results suggest that alpha phase gates visual responses during the first feed-forward sweep of processing.


title: Accounting for point count ambient noise increases population size estimates

abstract: Ambient noise is an integral component of natural environments, but it also creates challenges for avian monitoring programs. Ambient noise can mask bird vocalizations from observers during point counts, which may lead to systematic undercounting of birds in noisy environments. Here, we estimate detection probability and population size in models that either account for or omit the influence of ambient noise. We used data for four bird species, from 2228 point counts that were conducted during the 2019 Mountain Birdwatch field season. Community scientists assessed ambient noise using a simple scale. Despite relatively quiet conditions at sampling locations (x = 2.48), our information theoretical approach favored N-mixture models that incorporated ambient noise into the detection function for all four species. At the noisiest sampling locations, our models predicted detection probabilities that were as low as 10% for some species. Accounting for ambient noise resulted in a modest, mean increase of [≤]3.29% in the total population size for each species. Following our approach, other researchers can easily incorporate ambient noise assessments into their field protocols and analyses, with minimal costs or added complexity, to increase the comparability of studies conducted within different acoustic environments.


title: Autism risk gene POGZ promotes chromatin accessibility and expression of clustered synaptic genes

abstract: De novo mutations in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Here we find that in the developing mouse and human brain POGZ binds predominantly euchromatic loci and these are enriched for human neurodevelopmental disorder genes and transposable elements. We profile chromatin accessibility and gene expression in Pogz-/- mice and find that POGZ promotes chromatin accessibility of candidate regulatory elements (REs) and the expression of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with HP1{gamma} and ADNP, another high-confidence ASD risk gene. In Pogz+/- mice, Adnp expression is reduced. We postulate that reduced POGZ dosage disrupts cortical function through alterations in the POGZ-ADNP balance which modifies neuronal gene expression.


title: Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome

abstract: Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal excitation. In exploring the basis of these N-glycosylation alterations, we discovered they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed with neuronal excitation were in close association with ER exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway, or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrites satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction and disease.


title: Engineering paralog-specific PSD-95 synthetic binders as potent and minimally invasive imaging probes

abstract: Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.


title: Thunderstruck: The ACDC model of flexible sequences and rhythms in recurrent neural circuits

abstract: Adaptive sequential behavior is a hallmark of human cognition. In particular, humans can learn to produce precise spatiotemporal sequences given a certain context. For instance, musicians can not only reproduce learned action sequences in a context-dependent manner, they can also quickly and flexibly reapply them in any desired tempo or rhythm without overwriting previous learning. Existing neural network models fail to account for these properties. We argue that this limitation emerges from the fact that sequence information (i.e., the position of the action) and timing (i.e., the moment of response execution) are typically stored in the same neural network weights. Here, we augment a biologically plausible recurrent neural network of cortical dynamics to include a basal ganglia-thalamic module which uses reinforcement learning to dynamically modulate action. This "associative cluster-dependent chain" (ACDC) model modularly stores sequence and timing information in distinct loci of the network. This feature increases computational power and allows ACDC to display a wide range of temporal properties (e.g., multiple sequences, temporal shifting, rescaling, and compositionality), while still accounting for several behavioral and neurophysiological empirical observations. Finally, we apply this ACDC network to show how it can learn the famous "Thunderstruck" song intro and then flexibly play it in a "bossa nova" rhythm without further training.


title: Modulation of endometrial E-Cadherin and N-Cadherin by ovarian steroids and embryonic stimuli

abstract: The endometrium is a dynamic tissue that undergoes extensive remodelling to attain a receptive state which is further modulated in presence of an embryo for successful initiation of pregnancy. Cadherins are the proteins of junctional complex of which E-cadherin (E-Cad) is crucial for maintaining epithelial cell state and integrity of the epithelial barrier; gain of N-cadherin (N-Cad) in epithelial cells leads to epithelial to mesenchymal transition (EMT). In the present study, we aimed to investigate the expression of E-Cad and N-Cad in the mouse endometrial luminal epithelium and its modulation by estrogen, progesterone and embryonic stimuli. We observed that E-Cad is diffusely expressed in the luminal epithelium of mouse endometrium during the estrus stage and upon estrogen treatment. It is apico-laterally and basolaterally sorted at the diestrus stage and in response to combined treatment of estrogen and progesterone. In 3D spheroids of human endometrial epithelial cells, combined treatment with estrogen and progesterone led to lateral sorting of E-Cad. In the mouse endometrium at the time of embryo implantation, there is loss of E-Cad which was associated with the gain of N-Cad suggestive of EMT in the luminal epithelium. This EMT is possibly driven by embryonic stimuli as treatment with estrogen and progesterone did not lead to gain of N-Cad expression. In conclusion, the present study demonstrates that steroid hormones directly affect E-Cad sorting in the endometrial epithelium.

category: developmental biology


title: The human GID complex engages two independent modules for substrate recruitment

abstract: The human GID (hGID) complex is an evolutionary conserved E3 ubiquitin ligase regulating diverse biological processes including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi-subunit complex remains poorly understood. While the yeast GID complex recognizes Pro/N-end rule substrates via yeast Gid4, the human GID complex requires a WDR26/Gid7-dependent module to trigger proteasomal degradation of mammalian HBP1. Here, using biochemical assays, crosslinking-mass spectrometry and cryo-electron microscopy, we show that hGID unexpectedly engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 together with RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N-end rule degron. GID4 substrate binding and ligase activity is regulated by ARMC8, while the shorter ARMC8{beta} isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo-EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring-like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non-overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provide insights into the regulation of its ligase activity.


title: Label-free tracking and massmeasurement of single proteins onlipid bilayers

abstract: We introduce dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of membrane-associated proteins. Our method enables quantitative studies of their mobility, membrane affinity and interactions at the single molecule level. Application to the membrane remodelling GTPase dynamin1 reveals heterogeneous mixtures of oligomers suggesting that the fundamental building block for oligomerisation is a dimer, challenging current tetramer-centric models. Dynamic mass photometry has the ability to transform our approach to studying biomolecular mechanisms in and on lipid bilayers.

category: biophysics


title: Mass-sensitive particle tracking (MSPT) to elucidate the membrane-associated MinDE reactioncycle

abstract: In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. So far, it has been consensus that their observation with sufficient sensitivity and time resolution requires the introduction of - predominantly fluorescent-labels to the system. However, the recent advent of mass photometry to quantify mass distributions of unlabelled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, we introduce mass-sensitive particle tracking (MSPT), enabling simultaneous label-free tracking and monitoring of molecular masses of single biomolecules diffusing on lipid membranes. We applied this approach to the highly non-linear reaction cycles underlying MinDE protein self-organisation. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. We found that MinD assembles into complexes larger than the commonly postulated dimer, through lateral interactions of membrane-bound complexes and subunit recruitment from solution. Furthermore, the ATPase-activating protein MinE interconnects MinD into high-molecular-weight heteromeric complexes and affects their subunit turnover and concerted membrane release. This study demonstrates the potential of MSPT to enhance our quantitative understanding of both prokaryotic and eukaryotic membrane-associated biological systems.


title: Thermal stress induces positive phenotypic and molecular feedback loops in zebrafish embryos

abstract: Aquatic organisms must cope with both rising and rapidly changing temperatures. These environmental changes can affect numerous traits, from molecular to ecological scales. Biotic stressors can induce the release of chemical cues which trigger behavioural responses in other individuals. In this study, we infer whether abiotic stressors, such as fluctuating temperature, may similarly propagate stress responses between individuals in fish not directly exposed to the stressor. To test this hypothesis, zebrafish (Danio rerio) embryos were exposed for 24 hours to fluctuating thermal stress, to medium in which another embryo was thermally stressed before ("stress medium"), and to a combination of these. Growth, behaviour, and expression of a panel of genes were used to characterise the thermal stress response and its propagation between embryos. Both high temperatures and stress medium significantly accelerated development and altered embryonic behaviour. Thermal stress significantly decreased the expression of the antioxidant gene SOD1, eight hours after the end of exposure. Of note, we found that the expression of sulfide:quinone oxidoreductase (SQOR), likewise a part of the antioxidant metabolism relevant in vertebrate stress response, and of interleukin-1{beta} (IL-1{beta}), involved in the immune response, were significantly altered by stress medium. This study illustrates the existence of positive thermal stress feedback loops in zebrafish embryos that induce stress in conspecifics. This evidence that thermal stress due to fluctuating, high temperatures can be propagated may be relevant for species found in high densities, either in aquaculture or in the natural environment, in a context of global change.

category: physiology


title: Caspase-dependent cleavage of DDX21 suppresses host innate immunity

abstract:
DEAD (Glu-Asp-Ala-Glu)-box RNA helicases have been proven to contribute to antiviral innate immunity. DDX21 RNA helicase was identified as a nuclear protein involved in ribosomal RNA processing and RNA unwinding. DDX21 was also proved to be the scaffold protein in the complex of DDX1-DDX21-DHX36 which senses double strand RNA and initiates downstream innate immunity. Here, we identified that DDX21 undergoes caspase-dependent cleavage after virus infection and treatment with RNA/DNA ligands, especially for RNA virus and ligands. Caspase-3/6 cleave DDX21 at D126 and promotes its translocation from the nucleus to the cytoplasm in response to virus infection. The cytoplasmic cleaved DDX21 negatively regulates the IFN-{beta} signaling pathway by suppressing the formation of DDX1-DDX21-DHX36 complex. Thus, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immunity response towards virus.

ImportanceInnate immunity serves as the first barrier against virus infection. DEAD (Glu-Asp-Ala-Glu)-box RNA helicases, originally considered to be involved RNA processing and RNA unwinding, have been shown to play an important role in anti-viral innate immunity. The precise regulation of innate immunity is critical for the host because the aberrant production of cytokines leads to unexpected pathological consequences. Here, we identified DDX21 was cleaved at D126 by virus infection and treatment with RNA/DNA ligands via the caspase-3/6-dependent pathway. The cytoplasmic cleaved DDX21 negatively regulates the IFN-{beta} signaling pathway by suppressing the formation of DDX1-DDX21-DHX36 complex. In sum, our data identify DDX21 as a regulator of immune balance and most importantly uncover a potential role of DDX21 cleavage in the innate immunity response towards virus.

category: microbiology


title: Bacterioplankton diversity and pollution levels in the estuarine regions of Chaliyar and Anjarakkandi rivers, Kerala, India

abstract: Rivers and the estuarine regions play crucial roles in the environment by controlling the nutrient cycling, waste disposal, and also in supporting biodiversity. However, the estuarine regions of rivers are highly susceptible to disruptive changes by anthropogenic pollutants and eutrophication. Bacterioplankton diversity is the best indicators of the pollution level and eutrophication status of the rivers. The present study evaluated the eutrophication status and bacterioplankton diversity of the estuarine regions of Chaliyar and the Anjarakkandi originated from the Western Ghats. The present study observed the presence of increased incidence of bacterioplankton comprising of proteobacteria, actinobacteria and cyanobacteria in the estuarine regions of Anjarakkandi river compared to that of Chaliyar river. Further, the percentage incidence of faecal bacteria including Bacillus subtilis and E. coli, were also found to be in high in the Anjarakkandi river; in corroborating with this, the diversity of nitrogen metabolizing bacteria was also higher in the estuarine samples of Anjarakkandi river. Corroborating with this, the levels of water nutrients including nirates, nitrites and phosphates were high in the Anjarakkandi compared to the Chaliyar river. Together, it is possible that there might be increased faecal contamination and subsequent increased eutrophication in the estuarine regions of Anjarakkandi river. Altogether, the study concludes that the Anjarakkandi river may be more polluted and which is partly contributed by faecal contaminants and also responsible for the change in the bacterioplankton community of the estuaries.