Miracle-dz/UniFAR-FTUs huggingface dataset:
Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
Lipid kinases VPS34 and PIKfyve coordinate a phosphoinositide cascade to regulate retriever-mediated recycling on endosomes

Cell-surface receptors control how cells respond to their environment. Many cell-surface receptors recycle from endosomes to the plasma membrane via a recently discovered pathway, which includes sorting-nexin SNX17, Retriever, WASH and CCC complexes. Here we discover that PIKfyve and its upstream PI3-kinase VPS34 positively regulate this pathway. VPS34 produces PI3P, which is the substrate for PIKfyve to generate PI3,5P2. We show that PIKfyve controls recycling of cargoes including integrins, receptors that control cell migration. Furthermore, endogenous PIKfyve colocalizes with SNX17, Retriever, WASH and CCC complexes on endosomes. Importantly, PIKfyve inhibition causes a loss of Retriever and CCC from endosomes, and mutation of the lipid binding site on a CCC subunit impairs its endosomal localization and delays integrin recycling. In addition, we show that recruitment of SNX17 is an early step and requires VPS34. These discoveries suggest that VPS34 and PIKfyve coordinate an ordered pathway to regulate recycling from endosomes and suggest how PIKfyve functions in cell migration.
Correlated Drug Action as a Baseline Model for Combination Therapy in Patient Cohorts and Cell Cultures

Identifying and characterizing the effect of combination therapies is of paramount importance in various diseases, including cancer. Various competing null models have been proposed to serve as baselines against which to compare the effect of drug combinations. In this work, we introduce Correlated Drug Action (CDA), a baseline model for the study of drug combinations in both cell cultures and in patient populations. CDA assumes that the efficacy of pairs of drugs to be used in a combination may be correlated, that is, if the efficacy of a drug in a given patient or cell is high (low), then the efficacy of the other drug may also be high (low) in the same patient or cell. Our model can be used in the temporal domain (temporal CDA or tCDA) to explain survival curves in patient populations, and in the dose domain (dose CDA or dCDA), to explain dose-response curves in cell cultures. At the level of clinical trials, we demonstrate tCDA’s utility in identifying possibly synergistic combinations and cases where the combination can be explained in terms of the monotherapies. At the level of cells in culture, dCDA generalizes null models such as Bliss independence, the Highest Single Agent model, the dose equivalence principle, and is consistent with what should be expected in sham combinations. We demonstrate the applicability of dCDA in assessing combinations in experimental MCF7 cell-line data by introducing a new metric, the Excess over CDA (EOCDA).
ProteinBERT: a universal deep-learning model of protein sequence and function

Self-supervised deep language modeling has shown unprecedented success across natural language tasks, and has recently been repurposed to biological sequences. However, existing models and pretraining methods are designed and optimized for text analysis. We introduce ProteinBERT, a deep language model specifically designed for proteins. Our pretraining scheme consists of masked language modeling combined with a novel task of Gene Ontology (GO) annotation prediction. We introduce novel architectural elements that make the model highly efficient and flexible to very large sequence lengths. The architecture of ProteinBERT consists of both local and global representations, allowing end-to-end processing of these types of inputs and outputs. ProteinBERT obtains state-of-the-art performance on multiple benchmarks covering diverse protein properties (including protein structure, post translational modifications and biophysical attributes), despite using a far smaller model than competing deep-learning methods. Overall, ProteinBERT provides an efficient framework for rapidly training protein predictors, even with limited labeled data. Code and pretrained model weights are available at https://github.com/nadavbra/protein_bert.
Assembly and levels of P-TEFb depend on reversible phosphorylation of cyclinT1

The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, it is absent in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions require phosphorylation of two threonine residues (Thr143 and Thr149) in CycT1 by PKC. Conversely, PP1 dephosphorylates these sites. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminal differentiation of cells.
Building the cytokinetic contractile ring in an early embryo: Initiation as clusters of myosin II, anillin and septin, and visualization of a septin filament network

The cytokinetic contractile ring (CR) was first described some 50 years ago, however our understanding of the assembly and structure of the animal cell CR remains incomplete. We recently reported that mature CRs in sea urchin embryos contain myosin II mini-filaments organized into aligned concatenated arrays, and that in early CRs myosin II formed discrete clusters that transformed into the linearized structure over time. The present study extends our previous work by addressing the hypothesis that these myosin II clusters also contain the crucial scaffolding proteins anillin and septin, known to help link actin, myosin II, RhoA, and the membrane during cytokinesis. Super-resolution imaging of cortices from dividing embryos indicates that within each cluster, anillin and septin2 occupy a centralized position relative to the myosin II mini-filaments. As CR formation progresses, the myosin II, septin and anillin containing clusters enlarge and coalesce into patchy and faintly linear patterns. Our super-resolution images provide the initial visualization of anillin and septin nanostructure within an animal cell CR, including evidence of a septin filament network. Furthermore, Latrunculin-treated embryos indicated that the localization of septin or anillin to the myosin II clusters in the early CR was not dependent on actin filaments. These results highlight the structural progression of the CR in sea urchin embryos from an array of clusters to a linearized purse string, the association of anillin and septin with this process, and provide, for the first time, the visualization of septin filament higher order structure in an animal cell CR.
Nonsense-mediated mRNA decay uses complementary mechanisms to suppress mRNA and protein accumulation

A reporter system for quantitative measurements of NMD-sensitive mRNA and protein levels in mammalian cells shows that NMD suppresses protein levels to a greater degree than RNA levels. Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.
Intra-dorsal striatal acetylcholine M1 but not dopaminergic D1 or glutamatergic NMDA receptor antagonists inhibit consolidation of duration memory in interval timing

The striatal beat frequency (SBF) model assumes that striatal medium spiny neurons encode duration via synaptic plasticity. Muscarinic 1 (M1) cholinergic receptors, as well as dopamine and glutamate receptors, are important for neural plasticity in the dorsal striatum. Therefore, we investigated the effect of inhibiting these receptors on the formation of duration memory. After sufficient training in a Peak interval (PI)-20 s procedure, rats were given a single or mixed infusion of a selective antagonist for the dopamine D1 receptor (SCH23390, 0.5 µg per side), the NMDA-type glutamate receptor (D-AP5, 3 µg), or the M1 receptor (pirenzepine, 10 µg) bilaterally in the dorsal striatum, immediately before starting a PI 40 s session (shift session). On the next day, the rats were tested for new duration memory (40 s) in a session in which no lever presses were reinforced (probe session). In the shift session, performance was tie, irrespective of the drug injected. However, in the probe session, the mean peak time (an index of duration memory) of the M1 + NMDA co-blockade group, but not of the D1 + NMDA co-blockade group, was lower than that of the control group (Exp. 1 and 2). In Exp. 3, the effect of the co-blockade of M1 and NMDA receptors was replicated. Moreover, sole blockade of M1 receptors induced the same effect as M1 and NMDA blockade. These results suggest that in the dorsal striatum, the M1 receptor, but not the D1 or NMDA receptors, are involved in the consolidation of duration memory. Highlights D1 and NMDA receptors are not involved in the formation of duration memory. M1 receptors are involved in the consolidation of duration memory. These findings narrow the candidates for the receptor types inducing neuronal plasticity underlying the formation of duration memory in the SBF model.
Functional characterization of RELN missense mutations involved in recessive and dominant forms of Neuronal Migration Disorders

RELN is a large secreted glycoprotein that acts at multiple steps of cerebral cortex development, including neuronal migration. Only recessive mutations of the Reelin gene (RELN) have been associated with human cortical malformations and none has been functionally characterized. We identified novel missense RELN mutations in both compound and de novo heterozygous patients exhibiting an array of neuronal migration disorders (NMDs) as diverse as pachygyria, polymicrogyria and heterotopia. Most mutations caused defective RELN secretion in vitro and, when ectopically expressed in the embryonic mouse cortex, affected neuronal aggregation and/or migration in vivo. We determined the de novo heterozygous mutations acted as dominant negative and demonstrated that RELN mutations mediate not only recessive, but also dominant NMDs. This work assesses for the first time the pathogenicity of RELN mutations showing a strong genotype-phenotype correlation. In particular, the behavior of the mutant proteins in vitro and in vivo predicts the severity of cortical malformations and provides valuable insight into the pathogenesis of these disorders.
Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated1,2. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens1. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs3–5 (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth6–8, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.
Diversity of satellite glia in sympathetic and sensory ganglia

Satellite glia are the major glial type found in ganglia of the peripheral nervous system and wrap around cell bodies of sympathetic and sensory neurons that are very diverse. Other than their close physical association with peripheral neurons, little is known about this glial population. Here, we performed single cell RNA sequencing analysis and identified five different populations of satellite glia from sympathetic and sensory ganglia. We identified three shared populations of satellite glia enriched in immune-response genes, immediate-early genes and ion channels/ECM-interactors, respectively. Sensory- and sympathetic-specific satellite glia are differentially enriched for modulators of lipid synthesis and metabolism. Sensory glia are also specifically enriched for genes involved in glutamate turnover. Further, satellite glia and Schwann cells can be distinguished by unique transcriptional signatures. This study reveals remarkable heterogeneity of satellite glia in the peripheral nervous system.
Efficient Alcoholic Conversion of Glycerol by Engineered Saccharomyces cerevisiae

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae hinders its use in these methods. Here, we have aimed to develop a complete strategy for the generation of efficient glycerol-converting yeast by modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O2-dependent dynamic shuttle, while abolishing both glycerol phosphorylation and biosynthesis. By following a vigorous glycerol oxidation pathway, the engineered strain increased the conversion efficiency (CE) to up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate > 1 g·L·h, when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain fermented a mixture of glycerol and glucose, producing > 86 g/L bioethanol with 92.8% CE. To our knowledge, this is the highest ever reported titer in this field. Notably, this strategy changed conventional yeast from a non-grower on minimal medium containing glycerol to a fermenting strain with productivity of 0.25−0.5 g·L·h and 84−78% CE, which converted 90% of the substrate to products. Our findings may improve the utilization of glycerol in several eco-friendly biorefinery approaches. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Co-conversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, titer productivity hinders the industrial application of this process. Therefore, generation of efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Furthermore, glycerol is an important carbon source for the production of various chemicals. Hence, control of the metabolic flux of yeast grown on glycerol will contribute to the generation of cell factories that produce chemicals such as ethanol from glycerol, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.
Tribological behavior of diesel/biodiesel blend from waste cooking oil and ethanol

ABSTRACT One kind of novel biodiesel waste cooking oil ethyl ester (WCOEE) was prepared via transesterfication reaction between waste cooking oil and ethanol. The tribological behavior of diesel/WCOEE blend was evaluated with a four-ball tribometer. The wear resistance, extreme pressure, and friction reduction of the blend were improved with increasing WCOEE. The optimal content of WCOEE in the blend was 20 vol%. It was also found that free fatty acids (FFAs) had a positive effect on the wear resistance of blend. The lubrication improvement of the blend was ascribed to the formation of polyester film and high polarity of fatty acid ethyl ester.
The recombinase protein is a torque sensitive molecular switch

How a nano-searcher finds its nano-target is a general problem in non-equilibrium statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies are paired, that intact homologous sequence serves as a template to reconstitute the damaged DNA sequence, enabling the cell to survive without genetic mutations. To succeed, the search must stop only when the perfect homology is found. The biological process that ensures such a genomic integrity is called Homologous Recombination and is promoted by the Recombinase proteins. In this article, we use torque-sensitive magnetic tweezers to measure the free-energy landscape of the human Recombinase hRad51 protein assembled a DNA fragment. Based on our measurements we model the hRad51/DNA complex as an out-of-equilibrium two-state system and provide a thermodynamical description of Homologous Recombination. With this dynamical two-state model, we suggest a mechanism by which the recombinase proteins discriminate between homologous and a nonhomologous sequences.
Local Structure and L1- and L3-Edge X-ray Absorption Near Edge Structure of Late Lanthanide Elements (Ho, Er, Yb) in Their Complex Oxides

Characteristic features of Ho, Er, and Yb L1-edge and L3-edge XANES spectra of their various complex oxides were investigated to find a relationship to the local configuration around these late lanthanide atoms. A pre-edge peak area of the L1-edge or a full width at half-maximum of the white line of the L3-edge XANES spectra have a significant correlation with an abstract geometrical index defined by bond angles formed by a center atom, Ho, Er, or Yb, and two adjacent oxygen atoms, which acts as an indicator of disorder of the local configuration. Theoretical calculation based on multiple scattering theory revealed a general trend of the pre-edge peak at Ln L1-edge XANES spectra and the broadening of the white line of Ln L3-edge XANES spectra. This finding will open a way to analyze the local structure of late lanthanide elements by means of XANES spectroscopy.
Transfusion Reactions in Pediatric Patients: An Analysis of 5 Years of Hemovigilance Data From a National Center for Children's Health in China

Objective: This study aimed to describe transfusion reactions of pediatric patients from a National Center for Children's Health in China and to examine reaction incidents, reaction types by blood transfusion, and the associated blood products resulting in transfusion reactions. Methods: We compared transfusion reaction rates, among platelets, plasma, and red blood cells (RBCs) using a retrospective analysis of pediatric patients treated with blood transfusion based on data from the National Center for Children's Health (Beijing, China) by a hemovigilance reporting system from January 2015 to December 2019. Results: Over the past 5 years, 165 reactions were reported, and the overall incidence was 1.35‰ (95% CI: 1.14–1.55‰; 165/122,652); for each separate year, the incidences were 1.25‰ (95% CI: 0.76–1.74‰; 25/20,035; 2015), 1.09‰ (95% CI: 0.65–1.52‰; 24/22,084; 2016), 1.66‰ (95% CI: 1.14–2.18‰; 39/23,483; 2017), 1.36‰ (95% CI: 0.92–1.81‰; 36/26,440; 2018) and 1.34‰ (95% CI: 0.93–1.75‰; 41/30,610; 2019). Transfusion reaction incidents by person included 0.37‰ (95% CI: 0.21–0.53‰; 21/56,815) RBCs, 2.98‰ (95% CI: 2.33–3.64‰; 79/26,496) platelets and 1.65‰ (95% CI: 1.25–2.05‰; 65/39,341) frozen plasma. According to the analysis by blood products, the incidence of transfusion was 0.34‰ (95% CI: 0.20–0.48‰; 23/66,958) for RBCs, 3.21‰ (95% CI: 2.50–3.92‰; 78/24,318.5) for platelets, and 0.94‰ (95% CI: 0.71–1.17‰; 64/67,912) for frozen plasma. Transfusion reactions were most commonly associated with platelets, followed by plasma and RBC transfusions. The types of blood transfusion reactions were mainly allergic reactions (86.67%) and febrile non-hemolytic transfusion reactions (FNHTRs, 4.24%). The disease types of pediatric patients with transfusion reactions were concentrated among those with blood system diseases. A total of 80.61% of children with transfusion reactions had a previous blood transfusion history. Conclusions: Transfusion reactions are still relatively common in pediatric patients, and additional studies are necessary to address the differences in reaction rates, especially allergic and FNHTRs. Robust hemovigilance systems do include a special section dedicated to children will further the understanding of these reactions and trends, and prospective randomized clinical controlled trials may need to be conducted to perform preventive and corrective measures.