bluuebunny/crossref_metadata_2025 huggingface dataset:
Heavy metals in moss guide environmental justice investigation: a case study using community science in Seattle, WA, USA
Authors: Jovan Sarah E. , Zuidema Christopher , Derrien Monika M. , Bidwell Amanda L. , Brinkley Weston , Smith Robert J. , Blahna Dale , Barnhill Roseann , Gould Linn , Rodríguez Alberto J. , Amacher Michael C. , Abel Troy D. , López Paulina
Published: May 2022

Heavy metals concentrations often vary at small spatial scales not captured by air monitoring networks, with implications for environmental justice in industrial-adjacent communities. Pollutants measured in moss tissues are commonly used as a screening tool to guide use of more expensive resources, like air monitors. Such studies, however, rarely address environmental justice issues or involve the residents and other decision-makers expected to utilize results. Here, we piloted a community science approach, engaging over 55 people from nine institutions, to map heavy metals using moss in two industrial-adjacent neighborhoods. This area, long known for disproportionately poor air quality, health outcomes, and racial inequities, has only one monitor for heavy metals. Thus, an initial understanding of spatial patterns is critical for gauging whether, where, and how to invest further resources towards investigating heavy metals. Local youth led sampling of the moss Orthotrichum lyellii from trees across a 250×250-m sampling grid (n = 79) and generated data comparable to expert-collected samples (n = 19). We mapped 21 chemical elements measured in moss, including 6 toxic ‘priority’ metals: arsenic, cadmium, chromium, cobalt, lead, and nickel. Compared to other urban O. lyellii studies, local moss had substantially higher priority metals, especially arsenic and chromium, encouraging community members to investigate further. Potential hotspots of priority metals varied somewhat but tended to peak near the central industrial core where many possible emissions sources, including legacy contamination, converge. Informed by these findings, community members successfully advocated regulators for a second study phase – a community-directed air monitoring campaign to evaluate residents’ exposure to heavy metals – as is needed to connect moss results back to the partnership’s core goal of understanding drivers of health disparities. This follow-up campaign will measure metals in the PM10 fraction owing to clues in the current study that airborne soil and dust may be locally important carriers of priority metals. Future work will address how our approach combining bioindicators and community science ultimately affects success addressing longstanding environmental justice concerns. For now, we illustrate the potential to co-create new knowledge, to help catalyze and strategize next steps, in a complex air quality investigation.
Evolutionary investigations of the biosynthetic diversity in the skin microbiome usinglsaBGC
Authors: Salamzade Rauf , Cheong J.Z. Alex , Sandstrom Shelby , Swaney Mary Hannah , Stubbendieck Reed M. , Starr Nicole Lane , Currie Cameron R. , Singh Anne Marie , Kalan Lindsay R.
Published: May 2022

We developedlsaBGC, a bioinformatics suite that introduces several new methods to expand on the available infrastructure for genomic and metagenomic-based comparative and evolutionary investigation of biosynthetic gene clusters (BGCs). Through application of the suite to four genera commonly found in skin microbiomes, we uncover multiple novel findings on the evolution and diversity of their BGCs. We show that the virulence associated carotenoid staphyloxanthin inStaphylococcus aureusis ubiquitous across theStaphylococcusgenus but has largely been lost in the skin-commensal speciesStaphylococcus epidermidis. We further identify thousands of novel single nucleotide variants (SNVs) within BGCs from theCorynebacterium tuberculostearicumsp. complex, which we describe here to be a narrow, multi-species clade that features the most prevalentCorynebacteriumin healthy skin microbiomes. Although novel SNVs were approximately ten times as likely to correspond to synonymous changes when located in the top five percentile of conserved sites,lsaBGCidentified SNVs which defied this trend and are predicted to underlie amino acid changes within functionally key enzymatic domains. Ultimately, beyond supporting evolutionary investigations,lsaBGC provides important functionalities to aid efforts for the discovery or synthesis of natural products.
Strong basal/tonic TCR signals are associated with negative regulation of naive CD4+ T cells
Authors: Zinzow-Kramer Wendy M. , Eggert Joel , Scharer Christopher D. , Au-Yeung Byron B.
Published: May 2022

Tonic TCR signaling occurs constitutively in response to self-peptides presented by MHC (pMHC). Tonic TCR signal intensity correlates with Nur77-GFP reporter transgene expression. A broad range of Nur77-GFP is first detectable in post-selection thymocytes and persists in mature T cells. Nur77-GFPHI Ly6C− CD4+ T cells experience the strongest tonic TCR signaling and exhibit functional hypo-responsiveness to foreign pMHC stimulation. Gene expression analyses suggest similarities between the programs induced by strong tonic TCR signaling and T cell activation. However, the strongest tonic TCR signals also appear to induce expression of negative regulators, including coinhibitory receptors. Analysis of chromatin accessibility similarly suggest that strong tonic TCR signaling correlates with differentially higher accessibility of over 3000 chromatin regions in or near genes that encode positive and negative regulators of T cell activation. We propose that very strong tonic TCR signaling induces mechanisms of negative feedback to recalibrate T cell sensitivity.
Yield-Stress and Creep Control Depot Formation and Persistence of Injectable Hydrogels Following Subcutaneous Administration
Authors: Jons Carolyn K. , Grosskopf Abigail K. , Baillet Julie , Yan Jerry , Klich John H. , Appel Eric A.
Published: May 2022

Hydrogels that can be injected into the body using standard needles or catheters enable a minimally invasive strategy to prolong local delivery of therapeutic drug and cellular cargo. In particular, physically crosslinked hydrogels exhibit shear-thinning and self-healing behaviors enabling facile injectability and depot formation upon administration. While prior efforts to characterize these systems have focused on injectability and cargo release behaviors, prediction of cargo release in the body often assumes the materials form a depot rather than spreading out upon administration. Here, we evaluate how hydrogel rheology correlates with depot formation and persistence following subcutaneous administration in mice with two physicochemically-distinct, physically crosslinked hydrogel systems. We evaluate calcium-alginate and polymer-nanoparticle hydrogel systems exhibiting variable mechanical behaviors across several rheological properties (stiffness, viscoelasticity, yield stress, and creep). By relating measured rheological properties to depot formation and persistence time following subcutaneous administration, we identify that yield stress is predictive of initial depot formation while creep is predictive of depot persistence. Indeed, only materials with yield stresses greater than 25 Pa form robust depots and reduced creep correlates with longer depot persistence. These findings provide predictive insights into design considerations for hydrogel technologies capable of extended controlled release of therapeutic cargo.
In situ polyadenylation enables spatial mapping of the total transcriptome
Authors: McKellar David W. , Mantri Madhav , Hinchman Meleana , Parker John S.L. , Sethupathy Praveen , Cosgrove Benjamin D. , De Vlaminck Iwijn
Published: May 2022

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We apply this Spatial Total RNA-Sequencing (STRS) approach to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration, and highlight host transcriptional responses associated with local viral RNA abundance. In situ polyadenylation requires the addition of only a single step to a widely used protocol for spatial RNA-sequencing, and thus could be broadly and quickly adopted. Spatial RNA-sequencing of the total transcriptome will enable new insights into spatial gene regulation and biology.
Lung spatial profiling reveals a T cell signature in COPD patients with fatal SARS-CoV-2 infection
Authors: Yang Chen Xi , Tomchaney Michael , Landecho Manuel F. , Zamacona Borja R. , Oto Marta Marin , Zulueta Javier , Malo Joshua , Knoper Steve , Contoli Marco , Papi Alberto , Vasilescu Dragoş M. , Sauler Maor , Straub Christof , Tan Cheryl , Martinez Fernando D. , Bhattacharya Deepta , Rosas Ivan O. , Kheradmand Farrah , Hackett Tillie-Louise , Polverino Francesca
Published: May 2022

Rationale

People with pre-existing lung diseases like chronic obstructive pulmonary disease (COPD) are more likely to get very sick from SARS-CoV-2 disease 2019 (COVID-19), but an interrogation of the immune response to COVID-19 infection, spatial throughout the lung structure is lacking in patients with COPD.

Objectives

To profile the immune microenvironment of lung parenchyma, airways, and vessels of never- and ever-smokers with or without COPD, whom all died of COVID-19, using spatial transcriptomic and proteomic profiling.

Findings

The parenchyma, airways, and vessels of COPD patients, compared to control lungs had: 1) significant enrichment for lung resident CD45RO+ memory T cells; 2) downregulation of genes associated with T cell antigen-priming and memory T cell differentiation; 3) higher expression of proteins associated with SARS-CoV-2 entry and major receptor ubiquitously across the ROIs and in particular the lung parenchyma, despite similar SARS-CoV-2 structural gene expression levels.

Conclusions

The lung parenchyma, airways, and vessels of COPD patients have increased T-lymphocytes with a blunted memory T cell response and a more invasive SARS-CoV-2 infection pattern, and may underlie the higher death toll observed with COVID-19.
Artificial intelligence-based gas chromatography-olfactometry for sensory evaluation of key compounds in food ingredients
Authors: Shang Liang , Liu Chuanjun , Tang Fengzhen , Chen Bin , Liu Lianqing , Hayashi Kenshi
Published: May 2022

The use of an artificial intelligence (AI)-based prediction model of the structure-odor relationship (SOR) has shown great potential in the replacement of human panelists in gas chromatography-olfactometry (GCO). However, the Al-based GCO encounters issues such as poor accuracy, generalization, and practicality, owning to the insufficient feature extraction of odorant molecular structure. The purpose of this study is to the prediction of odor perception categories based on the odorant structure feature extraction by diverse deep neural networks, including molecular graphic convolution neural network (MG-CNN), molecular graph transformer neural network, and atom interaction neural networks. The results of the performance comparison of different feature extractors demonstrate that the MG-CNN model produces the highest accuracy and thus may be most suitable for the SOR prediction. It is hoped that the proposed method can be applied in practice as an auxiliary tool of GCO for the sensory evaluation of key compounds in food ingredients.

Highlights

Different deep neural networks were used to predict categorized odor descriptors.

End-to-end-based representation learning was performed for molecular feature extraction.

Molecular graphs with pre-training of convolution neural networks was most accurate.

Graphical Abstract
p38 MAPK Signaling Enhances Reovirus Replication by Facilitating Efficient Virus Entry, Virus Capsid Uncoating and Post-Uncoating Steps
Authors: Mohamed Adil , Shmulevitz Maya
Published: May 2022

Mammalian orthoreovirus serotype 3 Dearing an orphan human virus currently being pursued as an oncolytic virus in multiple phase I/II clinical trials. Previous clinical trials have emphasized the importance of patient pre-screening for prognostic markers to improve therapeutic success. However, currently, only generic cancer markers such as EGFR, Hras, Kras, Nras, Braf and/or p53 are utilized and have exhibited limited benefit in predicting therapeutic efficacy. Utilization of more specific markers that influence specific steps during reovirus replication could prove beneficial as prognostic markers. This study delineated the role of p38 MAPK signaling during reovirus infection and illustrated a connection between specific p38 MAPK isoforms and reovirus infection. Using a panel of specific p38 MAPK inhibitors and an inactive inhibitor analogue, we demonstrated that p38 MAPK signaling is essential for establishment of reovirus infection by enhancing reovirus endocytosis, facilitating efficient reovirus uncoating at the endo-lysosomal stage, and augmenting post uncoating replication steps. Using a broad panel of human breast cancer cell lines, we observed susceptibility of reovirus infection corresponded with virus binding and uncoating efficiency, which was strongly correlated with status of the p38β isoform. Together, our study proposes p38β as a potential prognostic marker for early stages of reovirus infection that are crucial to establishment of successful reovirus infection.
Strengthening vaccination delivery system resilience in the context of protracted humanitarian crisis: a realist-informed systematic review
Authors: Ismail Sharif A. , Lam Sze Tung , Bell Sadie , Fouad Fouad M. , Blanchet Karl , Borghi Josephine
Published: May 2022

Background

Childhood vaccination is among the most effective public health interventions available for the prevention of communicable disease, but coverage in many humanitarian settings is sub-optimal. This systematic review critically evaluated peer-review and grey literature evidence on the effectiveness of system-level interventions for improving vaccination coverage in protracted crises, focusing on how they work, and for whom, to better inform preparedness and response for future crises.

Methods

Realist-informed systematic review of peer-reviewed and grey literature. Keyword-structured searches were performed in MEDLINE, EMBASE and Global Health, CINAHL, the Cochrane Collaboration and WHOLIS, and grey literature searches performed through the websites of UNICEF, the Global Polio Eradication Initiative (GPEI) and Technical Network for Strengthening Immunization Services. Results were independently double-screened for inclusion on title and abstract, and full text. Data were extracted using a pre-developed template, capturing information on the operating contexts in which interventions were implemented, intervention mechanisms, and vaccination-related outcomes. Study quality was assessed using the MMAT tool. Findings were narratively synthesised.

Results

50 studies were included, most describing interventions applied in conflict or near-post conflict settings in sub-Saharan Africa, and complex humanitarian emergencies. Vaccination campaigns were the most commonly addressed adaptive mechanism (n=17). Almost all campaigns operated using multi-modal approaches combining service delivery through multiple pathways (fixed and roving), health worker recruitment and training and community engagement to address both vaccination supply and demand. Creation of collaterals through service integration showed generally positive evidence of impact on routine vaccination uptake by bringing services closer to target populations and leveraging trust that had already been built with communities. Robust community engagement emerged as a key unifying mechanism for outcome improvement across almost all of the intervention classes, in building awareness and trust among crisis-affected populations. Some potentially transformative mechanisms for strengthening resilience in vaccination delivery were identified, but evidence for these remains limited.

Conclusion

A number of interventions to support adaptations to routine immunisation delivery in the face of protracted crisis are identifiable, as are key unifying mechanisms (multi-level community engagement) apparently irrespective of context, but evidence remains piecemeal. Adapting these approaches for local system resilience-building remains a key challenge.
Can process mapping and a multi-site Delphi of perioperative professionals inform our understanding of system-wide factors that may impact operative risk?
Authors: Stubbs DJ , Bashford T , Gilder FJ , Nourallah B , Ercole A , Levy N , Clarkson PJ
Published: May 2022

Objectives

To examine whether the use of process mapping and a multidisciplinary Delphi can identify potential contributors to perioperative risk. We hypothesised that this approach may identify factors not represented in common perioperative risk tools and give insights of use to future research in this area.

Design

Multidisciplinary modified Delphi study

Setting

Two centres (one tertiary, one secondary) in the United Kingdom during 2020 amidst coronavirus pressures.

Participants

91 stakeholders from 23 professional groups involved in the perioperative care of older patients. Key stakeholder groups were identified through the use of process mapping of local perioperative care pathways.

Results

Response rate ranged from 51% in round one to 19% in round three. After round one, free text suggestions from the panel were combined with variables identified from perioperative risk scores. This yielded a total of 410 variables that were voted on in subsequent rounds. Including new suggestions from round two, 468/519 (90%) of the statements presented to the panel reached a consensus decision by the end of round three. Identified risk factors included patient level factors (such as ethnicity and socio-economic status); and organisational or process factors related to the individual hospital (such as policies, staffing, and organisational culture). 66/160 (41%) of the new suggestions did not feature in systematic reviews of perioperative risk scores or key process indicators. No factor categorised as ‘organisational’ is currently present in any perioperative risk score.

Conclusions

Through process mapping and a modified Delphi we gained insights into additional factors that may contribute to perioperative risk. Many were absent from currently used risk stratification scores. These results enable an appreciation of the contextual limitations of currently used risk tools and could support future research into the generation of more holistic datasets for the development of perioperative risk assessment tools.

Strengths and Weaknesses

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Novel use of process mapping to identify key perioperative stakeholders

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Multidisciplinary Delphi panel to gain breadth of perspective

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Performed across two sites

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Comprehensive results may be of use to other researchers designing perioperative research databases

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Results may be limited by low response rate in final round (although majority of consensus decisions made in round two)
COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds
Authors: Tappe Beeke , Lauruschkat Chris D. , Strobel Lea , Pantaleón García Jezreel , Kurzai Oliver , Rebhan Silke , Kraus Sabrina , Pfeuffer-Jovic Elena , Bussemer Lydia , Possler Lotte , Held Matthias , Hünniger Kerstin , Kniemeyer Olaf , Schäuble Sascha , Brakhage Axel A. , Panagiotou Gianni , White P. Lewis , Einsele Hermann , Löffler Jürgen , Wurster Sebastian
Published: May 2022

Patients suffering from coronavirus disease-2019 (COVID-19) are at high risk for deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis (CAPA) and COVID-19-associated mucormycosis (CAM). Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using anex-vivowhole blood (WB) stimulation assay, we challenged blood from twelve COVID-19 patients withAspergillus fumigatusandRhizopus arrhizusantigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakenedA. fumigatus- andR. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, WB from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response toA. fumigatusandR. arrhizusantigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients.
Vaccination as personal public-good provision
Authors: Reddinger J. Lucas , Charness Gary , Levine David
Published: May 2022

Vaccination against infectious diseases has both private and public benefits. We study whether social preferences—concerns for the well-being of other people—are associated with one’s decision regarding vaccination. We measure these social preferences for 549 online subjects with a public-good game and an altruism game. To the extent that one gets vaccinated out of concern for the health of others, contribution in the public-good game is analogous to an individual’s decision to obtain vaccination, while our altruism game provides a different measure of altruism, equity, and efficiency concerns. We proxy vaccine demand with how quickly a representative individual voluntarily took the initial vaccination for COVID-19 (after the vaccine was widely available). We collect COVID-19 vaccination history separately from the games to avoid experimenter-demand effects. We find a strong result: Contribution in the public-good game is associated with greater demand to voluntarily receive a first dose, and thus also to vaccinate earlier. Compared to a subject who contributes nothing, one who contributes the maximum ($4) is 58% more likely to obtain a first dose voluntarily in the four-month period that we study (April through August 2021). In short, people who are more pro-social are more likely to take a voluntary COVID-19 vaccination. Behavior in our altruism game does not predict vaccination. We recommend further research on the use of pro-social preferences to help motivate individuals to vaccinate for other transmissible diseases, such as the flu and HPV.
An anaphase switch in astral microtubule dynamics specifically requires the APC/CCdc20-dependent degradation of the mitotic cyclin Clb4
Authors: Zucca Federico , Li Jiaming , Visintin Clara , Gygi Steven P. , Visintin Rosella
Published: May 2022

Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar and astral microtubules (kMTs, iMTs and aMTs, respectively), among all aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, crucial for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4, provide a framework to understand aMT regulation in vertebrates and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.
Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability
Authors: Gorman Jason , Cheung Crystal Sao-Fong , Duan Zhijian , Sun Yaping , Wang Pengfei , Boyington Jeffrey C. , Biju Andrea , Bylund Tatsiana , Cheng Cheng , Ou Li , Stephens Tyler , Tsybovsky Yaroslav , Verardi Raffaello , Wang Shuishu , Yang Yongping , Zhang Baoshan , Zheng ChengYan , Zhou Tongqing , Mascola John R. , Ho David D. , Ho Mitchell , Kwong Peter D.
Published: May 2022

Lassa virus (LASV) is responsible for 100,000-300,000 zoonotic infections annually and poses a threat to public health. Development of antibody-based therapeutics or vaccines has been challenging because neutralizing antibodies – even among Lassa hemorrhagic fever survivors – are generally of low titer, and the target of neutralizing antibodies, the trimeric glycoprotein complex (GPC), a type 1-fusion machine with GP1 and GP2 subunits, has been difficult to produce. Here, we use structure-based design to obtain a soluble LASV GPC by engineering an inter-protomer disulfide (R207GCGP1-L326CGP2) and appending the T4-fibritin trimerization domain. We verified the antigenicity of this prefusion-stabilized LASV GPC against a panel of human antibodies and used electron microscopy (EM) to confirm its trimeric association. We panned the prefusion-stabilized LASV GPC against single domain ‘nanobody’ libraries and identified one of camel origin, which we named D5, which bound GPC with 27 nM affinity and neutralized the Josiah strain of LASV with an IC50 of 12 µg/ml when formatted into a bivalent IgG2a context. The cryo-EM structure of a ternary complex of the D5 nanobody, the antigen-binding fragment of human antibody 8.11G, and LASV GPC revealed D5 to recognize a site-of-vulnerability at the trimer apex. The recognized site appeared to be specific to GPC lacking cleavage of between GP1 and GP2 subunits. Collectively, our findings suggest that GPC-cleavage intermediates may be targets for LASV neutralization and define an apex-situated site of vulnerability for vaccine development.

Significance

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a biothreat to the world. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into requirements for antibody-mediated neutralization of LASV, we developed a prefusion-stabilized LASV glycoprotein trimer (GPC), panned it against phage libraries comprised single-domain antibodies or nanobodies from shark and camel, and identified one, D5, which – when placed into bivalent IgG2a context – could neutralize LASV. Cryo-EM analysis revealed D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. We propose this apex-situated site to be an attractive target for LASV vaccine and therapeutic development.
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

The 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 Summary

The 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

CASK 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

Emergence 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

The 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

Crosslinking 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

Ralstonia 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

Egg 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

The 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

People 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 Points

We 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

Recent 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

Altering 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

Transcriptional 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.