bluuebunny/crossref_metadata_2025_split huggingface dataset:
The PqsE active site as a target for small molecule antimicrobial agents againstPseudomonas aeruginosa
Authors: Taylor Isabelle R. , Jeffrey Philip D. , Moustafa Dina A. , Goldberg Joanna B. , Bassler Bonnie L. 
Published: May 2022

AbstractThe opportunistic pathogenPseudomonas aeruginosacauses antibiotic resistant, nosocomial infections in immuno-compromised individuals, and is a high priority for antimicrobial development. Key to pathogenicity inP. aeruginosaare biofilm formation and virulence factor production. Both traits are controlled by the cell-to-cell communication process called quorum sensing (QS). QS involves the synthesis, release, and population-wide detection of signal molecules called autoinducers. We previously reported that activity of the RhlR QS transcription factor depends on a protein-protein interaction with the hydrolase, PqsE, and PqsE catalytic activity is dispensable for this interaction. Nonetheless, the PqsE-RhlR interaction could be disrupted by substitution of an active site glutamate residue with tryptophan (PqsE(E182W)). Here, we show that disruption of the PqsE-RhlR interaction via either the E182W change or alteration of PqsE surface residues that are essential for the interaction with RhlR, attenuatesP. aeruginosainfection in a murine host. We use crystallography to characterize the conformational changes induced by the PqsE(E182W) substitution to define the mechanism underlying disruption of the PqsE-RhlR interaction. A loop rearrangement that repositions the E280 residue in PqsE(E182W) is responsible for the loss of interaction. We verify the implications garnered from the PqsE(E182W) structure using mutagenic, biochemical, and additional structural analyses. We present the next generation of molecules targeting the PqsE active site, including a structure of the tightest binding of these compounds, BB584, in complex with PqsE. The findings presented here provide insight for drug discovery againstP. aeruginosawith PqsE as the target.Author SummaryThe human pathogenPseudomonas aeruginosais resistant to many currently used antibiotics, making it a burden of urgent clinical importance.P. aeruginosapathogenicity is controlled by the bacterial cell-to-cell communication process called quorum sensing (QS). The function of one protein that controlsP. aeruginosaQS-directed virulence, RhlR, requires a protein-protein interaction with an enzyme called PqsE. When PqsE is blocked from interacting with RhlR,P. aeruginosais avirulent and incapable of infecting an animal host. Here, we validate the PqsE-RhlR interaction as a target for antibiotic development, and we present a mechanism for how such antibiotics could disrupt the PqsE-RhlR interaction. Discovery of new antibiotics would fulfill an unmet healthcare need by providing treatments to combatP. aeruginosainfections.
Regulation of Liprin-α phase separation by CASK is disrupted by a mutation in its CaM kinase domain
Authors: Tibbe Debora , Ferle Pia , Krisp Christoph , Nampoothiri Sheela , Mirzaa Ghayda , Assaf Melissa , Parikh Sumit , Kutsche Kerstin , Kreienkamp Hans-Jürgen 
Published: May 2022

AbstractCASK is a unique membrane associated guanylate kinase (MAGUK), due to its Ca2+/calmodulin-dependent kinase (CaMK) domain. We describe four male patients with a severe neurodevelopmental disorder with microcephaly carrying missense variants affecting the CaMK domain. One boy who carried the p.E115K variant and died at an early age showed pontocerebellar hypoplasia (PCH) in addition to microcephaly, thus exhibiting the classical MICPCH phenotype observed in individuals withCASKloss-of-function variants. All four variants selectively weaken the interaction of CASK with Liprin-α2, a component of the presynaptic active zone. Liprin-α proteins form spherical condensates in a process termed liquid-liquid phase separation (LLPS), which we observe here in Liprin-α2 overexpressing HEK293T cells and primary cultured neurons. Condensate formation is reversed by interaction of Liprin-α2 with CASK; this is associated with altered phosphorylation of Liprin-α2. The p.E115K variant fails to interfere with condensate formation. As the individual carrying this variant had the severe MICPCH disorder, we suggest that regulation of Liprin-α2-mediated LLPS is a new functional feature of CASK which must be maintained to prevent PCH.
Peptide derived nanobody inhibits entry of SARS-CoV-2 variants
Authors: Mendon Nivya , Ganie Rayees , Kesarwani Shubham , Dileep Drisya , Sasi Sarika , Lama Prakash , Chandra Anchal , Sirajuddin Minhajuddin 
Published: May 2022

AbstractEmergence of the new escape mutants of the SARS-CoV-2 virus has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS variants and other viruses in a short period of time becomes essential. Blocking the SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-ACE2 interaction has been already exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a 9 amino acid peptide from the receptor-binding motif (RBM) of Spike (S) protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the nanobody. In summary, our study presents a faster and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. This approach can also be widely extended to target other viruses and pathogens in the future.
SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid
Authors: Oliveira A. Sofia F. , Shoemark Deborah K. , Davidson Andrew D. , Berger Imre , Schaffitzel Christiane , Mulholland Adrian J. 
Published: May 2022

AbstractThe SARS-CoV-2 spike protein contains a fatty acid binding site, also found in some other coronaviruses (e.g. SARS-CoV), which binds linoleic acid and is functionally important. When occupied by linoleic acid, it reduces infectivity, by ‘locking’ the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. These simulations show that the fatty acid site is coupled to functional regions of the protein, some of them far from the site (e.g. in the receptor-binding motif, N-terminal domain, the furin cleavage site located in position 679-685 and the fusion peptide-surrounding regions) and identify the allosteric networks involved in these connections. Comparison of the response of the original (‘Wuhan’) spike with four variants: Alpha, Delta, Delta plus and Omicron BA.1 show that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the fatty acid site on Alpha are generally similar to the original protein, except for the receptor-binding motif and S71-R78 region which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629 and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, e.g. in differences in transmissibility and virulence. Experimental comparison of the effects of linoleic acid on different variants is warranted.
Xlink Mapping and AnalySis (XMAS) - Smooth Integrative Modeling in ChimeraX
Authors: Lagerwaard Ilse M. , Albanese Pascal , Jankevics Andris , Scheltema Richard A. 
Published: May 2022

AbstractCrosslinking mass spectrometry (XL-MS) adds tremendous value to structural biology investigations. Its main strength lies in uncovering structural information in the form of distance constraints between neighboring amino acids of proteins, protein regions and complex samples, which are difficult to assess by other analytical techniques. However, although several approaches have been proposed, interpreting XL-MS data in a structural context has been cumbersome. ChimeraX has gained momentum as a flexible and widely used software package for the visualization of structural data, but is currently lacking functionalities for integration of experimental XL-MS data. Here, we introduce XMAS, a bundle that allows users to load results from several XL-MS search engines directly into ChimeraX and map the information onto protein structures. Besides automatically locating distance constraints on protein structures, XMAS offers the possibility to work with replicate experiments and/or different crosslinkers, and filter this data based on the number of replicates for which a given distance constraint was detected, thereby increasing the data quality. Additionally, we introduce the concept of self-links, which allows easy modeling of homo-dimeric interactions. Its core functionality is extended by the implementation of seamless connections to the HADDOCK suite to streamline otherwise time-consuming tasks in structural modeling pipelines. We demonstrate these key elements of the XMAS bundle by modeling crosslinking data obtained from human fibrin clots. The software is freely available from the ChimeraX toolshed, with an extensive user manual and example datasets.
Diversity of Secretion System Apparatus in Tomato Wilt Causing Ralstonia solanacearum Strains: a Comparative Analysis Using in-silico Approach
Authors: Banerjee Goutam , Chatterjee Sandipan , Banerjee Pratik , Chattopadhyay Pritam 
Published: May 2022

AbstractRalstonia solanacearum (Rs) species is the leading cause of bacterial wilt disease in a wide range of host plants worldwide. In the present study, secretion system analysis of five tomato pathogenic Rs strains was carried out in-silico. This paper describes a new protocol to identify the secretion system components i.e. SSCs (T1SS-T6SS, Flg, T4P, and Tad-Tat). A total of 865 SSCs were identified using the new protocol. Contributions of SSCs into core-secretion system apparatus (i.e. SSA) were also studied. Synteny was discovered among the secretion system apparatus (SSA) where relative frequency of SSCs to core-SSA is high (>20%) which includes T1SS, T2SS, T5SS, T4P, and Tad-Tat, but excludes T3SS, T4SS, and Flg. To the best of our knowledge, this is the first report indicating that during the evolution of Rs, most of the secretion system apparatus (T1SS, T2SS, T5SS, T4P, and Tad-Tat) were highly conserved and came from a single ancestor, while T3SS and T6SS may have arrived later, probably from horizontal gene transfer.
Low temperature induced quiescence in parasitoids Trichogramma evanescens Westwood and Trichogramma chilonis Ishii reared on Plodia interpunctella (Hübner): Its utilization as quality control for mass rearing
Authors: Hasan Md. Mahbub , Parvin M. Nishat , Athanassiou Christos G. 
Published: May 2022

AbstractEgg parasitoid, Trichogramma spp. is an important potential biological control agent for wide range of lepidopteran pests. Cold storage of host eggs has been proposed as a valuable technique for ensuring the release of sufficient parasitoid numbers whenever it is needed. Thus, the impact of low temperatures to induce quiescence in Trichogramma evanescens and Trichogramma chilonis was studied using eggs of Plodia interpunctella as hosts. Prepupae of the parasitoids Trichogramma evanescens and T.chilonis were stored for 15, 30, 45, 60 and 75 d at 4°C, following a 7 d period of acclimation at 10°C. Both parasitoid species seem to survive unfavorable temperature conditions by entering a state of quiescence. Parasitism, adult emergence, female proportion relative to male and F1 adult emergence were not affected by cold storage in either parasitoid for up to 30 d of storage. Parasitized host eggs of P. interpunctella can be stored for up to 60 d at 4°C for both parasitoids, but there was no emergence at 75 d. Results clearly show that there are specific intervals of cold storage during which the parasitoids can remain unaffected for relatively long periods of time. Although we observed some adverse effect in longevity and parasitism rates, the technique described here can be further utilized in mass rearing strategies of egg parasitoids for relatively long periods that will allow shipment and application in biological control programs.
rTg(TauP301L)4510 mice exhibit increased VGLUT1 in hippocampal presynaptic glutamatergic vesicles and increased extracellular glutamate release
Authors: Taipala Erika , Pfitzer Jeremiah C. , Hellums Morgan , Reed Miranda , Gramlich Michael W. 
Published: May 2022

AbstractThe molecular pathways that contribute to the onset of symptoms in tauopathy models, including Alzheimer’s Disease (AD), are difficult to distinguish because multiple changes can happen simultaneously at different stages of disease progression. Understanding early synaptic alterations and their supporting molecular pathways is essential in order to develop better pharmacological targets to treat AD. Here we focus on an early onset rTg(TauP301L)4510 tauopathy mouse model that exhibits hyperexcitability in hippocampal neurons of adult mice that is correlated with presynaptic changes and increased extracellular glutamate levels. However, it is not clear if increased extracellular glutamate is caused by presynaptic changes alone, or if presynaptic changes are a contributing factor among other factors. To determine whether pathogenic tau alters presynaptic function and glutamate release, we studied cultured hippocampal neurons at 14-18 DIV from animals of both sexes to measure presynaptic changes in tauP301L positive mice. We observed that presynaptic vesicles exhibit increased vesicular glutamate transporter 1 (VGLUT1) using immunohistochemistry of fixed cells and an established pH-sensitive green fluorescent protein approach. We show that tauP301L positive neurons exhibit a 40% increase in VGLUT1 per vesicle compared to tauP301L negative littermates. Further, we use the extracellular glutamate reporter iGluSnFR to show that increased VGLUT1 per vesicle directly translates into a 40% increase in extracellular glutamate. Together, these results show that increased extracellular glutamate levels observed in tauP301L mice are not caused by increased vesicle exocytosis probability but rather are directly related to increased VGLUT1 transporters per synaptic vesicle.
HIV-linked gut dysbiosis associates with cytokine production capacity in viral-suppressed people living with HIV
Authors: Zhang Yue , Andreu-Sánchez Sergio , Vadaq Nadira , Wang Daoming , Matzaraki Vasiliki , van der Heijden Wouter , Gacesa Ranko , Weersma Rinse K , Zhernakova Alexandra , Vandekerckhove Linos , de Mast Quirijn , Joosten Leo A. B. , Netea Mihai G. , van der Ven Andre , Fu Jingyuan 
Published: May 2022

AbstractPeople living with HIV (PLHIV) are exposed to chronic immune dysregulation, even when virus replication is suppressed by antiretroviral therapy (ART). Given the emerging role of the gut microbiome in immunity, we hypothesized that the gut microbiome may be related to the cytokine production capacity of PLHIV. To test this hypothesis, we collected metagenomic data from 143 ART-treated PLHIV and assessed the ex vivo production capacity of eight different cytokines (IL-1β, IL-6, IL-1Ra, IL-10, IL17, IL22, TNF and IFN-γ) in response to different stimuli. We also characterized CD4+ T cell–counts, HIV reservoir and other clinical parameters. Compared to 190 age- and sex-matched controls and a second independent control cohort, PLHIV showed microbial dysbiosis that was correlated with viral reservoir levels, cytokine production capacity and sexual behavior. Notably, we identified two genetically different P. copri strains that were enriched in either PLHIV or healthy controls. The control-enriched strain was negatively associated with IL-10, IL-6 and TNF production, independent of age, sex and sexual behavior, and positively associated with CD4+ T cell–level, whereas the PLHIV-enriched strain showed no associations. Our findings suggest that modulating the gut microbiome may be a strategy to modulate immune response in PLHIV.Novel PointsWe identified compositional and functional changes in the gut microbiome of PLHIV that were strongly related to sexual behavior.HIV-associated bacterial changes are negatively associated with HIV reservoir. The relative abundance of Firmicutes bacterium CAG 95 and Prevotella sp CAG 5226 both show a negative association with CD4+ T cell–associated HIV-1 DNA.Prevotella copri and Bacteroides vulgatus show association with PBMC production capacity of IL-1β and IL-10 that is independent of age, sex, BMI and sexual behavior.We observed two genetically different P. copri strains that are enriched in PLHIV and healthy individuals, respectively.The control-related P. copri strain specifically shows a negative association with IL-10, IL-6 and TNF production and a positive association with CD4+ T cell–level. This suggests it plays a potential protective role in chronic inflammation, which may be related to enrichment of a specific epitope peptide.
A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice
Authors: Klioutchnikov Alexandr , Wallace Damian J. , Sawinski Juergen , Voit Kay-Michael , Groemping Yvonne , Kerr Jason N. D. 
Published: May 2022

AbstractRecent advances in head-mounted microscopes have enabled imaging of neuronal activity using genetic-tools in freely moving mice but these microscopes are restricted to recording in minimally lit arenas and imaging upper cortical layers. Here we built a 2 gram, three-photon excitation-based microscope, containing a z-drive that enabled access to all cortical layers while mice freely behaved in a fully lit environment. We show that neuronal population activity in cortical layer-4 and layer-6 was differentially modulated by lit and dark conditions during free exploration.
Dissection of a sensorimotor circuit that regulates aversion to odors and pathogenic bacteria inC. elegansby whole-brain simulation
Authors: Filipowicz Adam , Lalsiamthara Jonathan , Aballay Alejandro 
Published: May 2022

AbstractAltering behavior to reduce pathogen exposure is a key line of defense against pathogen attack for nearly all animals. The use ofCaenorhabditis elegansbacterial infection models have allowed for many insights into the molecular mechanisms of behavioral immunity. However, the neural circuitry between chemosensory neurons that sense pathogenic bacterial cues and the motor neurons responsible for avoidance-associated locomotion remains unknown. We found that backward locomotion was a component of learned pathogen avoidance, as animals pre-exposed toPseudomonas aeruginosaorEnterococcus faecalisshowed reflexive aversion to drops of the bacteria, requiring ASI, AWB, and AWC neurons and ASE, AWB, and AWC neurons, respectively. This response also involved intestinal distention and, forE. faecalis, required expression of TRPM channels in the intestine and excretory system. Using whole-brain simulation and functional assays, we uncovered a sensorimotor circuit governing learned reflexive aversion. This behavior is controlled by a four-layer neural circuit composed of olfactory neurons, interneurons, and motor neurons that control backward locomotion crucial for learned reflexive aversion to pathogenic bacteria, learned avoidance, and a repulsive odor. The discovery of a complete sensorimotor circuit for reflexive aversion demonstrates the utility of using theC. elegansconnectome and computational modeling in uncovering new neuronal regulators of behavior.
Reporter gene assays and chromatin-level assays define substantially non-overlapping sets of enhancer sequences
Authors: Lindhorst Daniel , Halfon Marc S. 
Published: May 2022

AbstractTranscriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence. We examine here whether these two types of definitions effectively identify the same sets of sequences and find that, concerningly, the overlap between the two groups is strikingly limited. Our results raise important questions as to the appropriateness of both old and new enhancer definitions.
The structure of the human LACTB filament reveals the mechanisms of assembly and membrane binding
Authors: Bennett Jeremy A. , Steward Lottie R. , Rudolph Johannes , Voss Adam P. , Aydin Halil 
Published: May 2022

AbstractMitochondria are complex organelles that play a central role in metabolism. Dynamic membrane-associated processes regulate mitochondrial morphology and bioenergetics in response to cellular demand. In tumor cells, metabolic reprogramming requires active mitochondrial metabolism for providing key metabolites and building blocks for tumor growth and rapid proliferation. To counter this, the mitochondrial serine beta-lactamase-like protein (LACTB) alters mitochondrial lipid metabolism and potently inhibits the proliferation of a variety of tumor cells. Mammalian LACTB is localized in the mitochondrial intermembrane space, where it assembles into filaments to regulate the efficiency of essential metabolic processes. However, the structural basis of LACTB polymerization and regulation remains incompletely understood. Here, we describe how human LACTB self-assembles into micron-scale filaments that increase their catalytic activity. The electron cryo-microscopy (cryoEM) structure defines the mechanism of assembly and reveals how highly ordered filament bundles stabilize the active state of the enzyme. We identify and characterize residues that are located at the filament-forming interface, and further show that mutations that disrupt filamentation reduce enzyme activity. Furthermore, our results provide evidence that LACTB filaments can bind lipid membranes. These data reveal the detailed molecular organization and polymerization-based regulation of human LACTB and provide new insights into the mechanism of mitochondrial membrane organization that modulates lipid metabolism.