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Found 35955 matches. Displaying 21-30
Xu SY, Campisi E, Li JQ, Fischetti VA
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Decontamination of Escherichia coli O157:H7 on fresh Romaine lettuce using a novel bacteriophage lysin

Raw vegetables are a key food for a healthy diet, but their increased consumption brings a higher risk for foodborne disease. Contamination of salad greens with Shiga toxin-producing Escherichia coli (STEC) O157:H7 has caused severe disease and important economic losses almost yearly in the United States over the last 10 years. To curb the risk of infections from contaminated produce, approaches based on bacterial virus - commonly known as bacteriophage or phage - have recently started to draw interest among other antimicrobial strategies. Phages enter bacterial cells to reproduce and cause cellular lysis to release their phage progeny at the end of their infection cycle. This lytic effect is caused by lysins, phage-encoded enzymes that have evolved to degrade the bacterial cell wall resulting in hypotonic lysis. When applied externally in their purified form, such enzymes are able to kill sensitive bacteria on contact in a similar way. Their unique bactericidal properties have made lysins effective antimicrobial agents in a variety of applications, from treating multidrug-resistant infections in humans to controlling bacterial contamination in several areas, including microbiological food safety. Here we describe a novel lysin, namely PlyEc2, with potent bactericidal activity against key gram-negative pathogens including E. coli, Salmonella, Shigella, Acinetobacter and Pseudomonas. PlyEc2 displayed high bactericidal activity against STEC to a concentration of 12.5 mu g/ml under different pH conditions. This lysin was also able to reduce the bacterial titer of several pathogenic strains in vitro by more than 5 logarithmic units, resulting in complete sterilization. Importantly, PlyEc2 proved to be a powerful produce decontamination agent in its ability to clear 99.7% of contaminating STEC O157:H7 in our Romaine lettuce leaf model. PlyEc2 was also able to eradicate 99.8% of the bacteria contaminating the washing solution, drastically reducing the risk of cross-contamination during the washing process. A sensory evaluation panel found that treatment with PlyEc2 did not alter the visual and tactile quality of lettuce leaves compared to the untreated leaves. Our study is the first to describe a highly effective lysin treatment to control gram-negative pathogenic contamination on fresh lettuce without the addition of membrane destabilizing agents.
Wieczorek M, Ti SC, Urnavicius L, Molloy KR, Aher A, Chait BT, Kapoor TM
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Biochemical reconstitutions reveal principles of human gamma-TuRC assembly and function

JOURNAL OF CELL BIOLOGY 2021 MAR 1; 220(3):? Article e202009146
The formation of cellular microtubule networks is regulated by the gamma-tubuhn ring complex (gamma-TuRC). This similar to 2.3 MD assembly of >31 proteins includes y-tubuhn and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB). The challenge of reconstituting the gamma-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human gamma-TuRC (gamma-TuRC-GFP) as a similar to 35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, gamma-TuRC(Delta LB)-GFP, which lacks MZT1 and actin. We show that gamma-TuRC(Delta LB)-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that gamma-TuRC-GFP resembles the native gamma-TuRC architecture, while gamma-TuRC(Delta LB)-GFP adopts a partial cone shape presenting only 8-10 gamma-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the gamma-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the holocomplex.
Vandana JJ, Lacko LA, Chen SB
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Phenotypic technologies in stem cell biology

CELL CHEMICAL BIOLOGY 2021 MAR 18; 28(3):257-270
The high-throughput phenotypic screen (HTPS) has become an emerging technology to discover synthetic small molecules that regulate stem cell fates. Here, we review the application of HTPS to identify small molecules controlling stem cell renewal, reprogramming, differentiation, and lineage conversion. Moreover, we discuss the use of HTPS to discover small molecules/polymers mimicking the stem cell extracellular niche. Furthermore, HTPSs have been applied on whole-animal models to identify small molecules regulating stem cell renewal or differentiation in vivo. Finally, we discuss the examples of the utilization of HTPS in stem cellbased disease modeling, as well as in the discovery of novel drug candidates for cancer, diabetes, and infectious diseases. Overall, HTPSs have provided many powerful tools for the stem cell field, which not only facilitate the generation of functional cells/tissues for replacement therapy, disease modeling, and drug screening, but also help dissect molecular mechanisms regulating physiological and pathological processes.
Novelli G, Liu J, Biancolella M, Alonzi T, Novelli A, Patten JJ, Cocciadiferro D, Agolini E, Colona VL, Rizzacasa B, Giannini R, Bigio B, Goletti D, Capobianchi MR, Grelli S, Mann J, McKee TD, Cheng K, Amanat F, Krammer F, Guarracino A, Pepe G, Tomino C, Tandjaoui-Lambiotte Y, Uzunhan Y, Tubiana S, Ghosn J, Notarangelo LD, Su HC, Abel L, Cobat A, Elhanan G, Grzymski JJ, Latini A, Sidhu SS, Jain S, Davey RA, Casanova JL, Wei WY, Pandolfi PP
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Inhibition of HECT E3 ligases as potential therapy for COVID-19

CELL DEATH & DISEASE 2021 MAR 24; 12(4):? Article 310
SARS-CoV-2 is responsible for the ongoing world-wide pandemic which has already taken more than two million lives. Effective treatments are urgently needed. The enzymatic activity of the HECT-E3 ligase family members has been implicated in the cell egression phase of deadly RNA viruses such as Ebola through direct interaction of its VP40 Protein. Here we report that HECT-E3 ligase family members such as NEDD4 and WWP1 interact with and ubiquitylate the SARS-CoV-2 Spike protein. Furthermore, we find that HECT family members are overexpressed in primary samples derived from COVID-19 infected patients and COVID-19 mouse models. Importantly, rare germline activating variants in the NEDD4 and WWP1 genes are associated with severe COVID-19 cases. Critically, I3C, a natural NEDD4 and WWP1 inhibitor from Brassicaceae, displays potent antiviral effects and inhibits viral egression. In conclusion, we identify the HECT family members of E3 ligases as likely novel biomarkers for COVID-19, as well as new potential targets of therapeutic strategy easily testable in clinical trials in view of the established well-tolerated nature of the Brassicaceae natural compounds.
Sharanowski BJ, Ridenbaugh RD, Piekarski PK, Broad GR, Burke GR, Deans AR, Lemmon AR, Lemmon ECM, Diehl GJ, Whitfield JB, Hines HM
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Phylogenomics of Ichneumonoidea (Hymenoptera) and implications for evolution of mode of parasitism and viral endogenization

Ichneumonoidea is one of the most diverse lineages of animals on the planet with >48,000 described species and many more undescribed. Parasitoid wasps of this superfamily are mostly beneficial insects that attack and kill other arthropods and are important for understanding diversification and the evolution of life history strategies related to parasitoidism. Further, some lineages of parasitoids within Ichneumonoidea have acquired endogenous virus elements (EVEs) that are permanently a part of the wasp's genome and benefit the wasp through host immune disruption and behavioral control. Unfortunately, understanding the evolution of viral acquisition, parasitism strategies, diversification, and host immune disruption mechanisms, is deeply limited by the lack of a robust phylogenetic framework for Ichneumonoidea. Here we design probes targeting 541 genes across 91 taxa to test phylogenetic relationships, the evolution of parasitoid strategies, and the utility of probes to capture polydnavirus genes across a diverse array of taxa. Phylogenetic relationships among Ichneumonoidea were largely well resolved with most higher-level relationships maximally supported. We noted codon use biases between the outgroups, Braconidae, and Ichneumonidae and within Pimplinae, which were largely solved through analyses of amino acids rather than nucleotide data. These biases may impact phylogenetic reconstruction and caution for outgroup selection is recommended. Ancestral state reconstructions were variable for Braconidae across analyses, but consistent for reconstruction of idiobiosis/koinobiosis in Ichneumonidae. The data suggest many transitions between parasitoid life history traits across the whole superfamily. The two subfamilies within Ichneumonidae that have polydnaviruses are supported as distantly related, providing strong evidence for two independent acquisitions of ichnoviruses. Polydnavirus capture using our designed probes was only partially successful and suggests that more targeted approaches would be needed for this strategy to be effective for surveying taxa for these viral genes. In total, these data provide a robust framework for the evolution of Ichneumonoidea.
Capoor MN, Konieczna A, McDowell A, Ruzicka F, Smrcka M, Jancalek R, Maca K, Lujc M, Ahmed FS, Birkenmaier C, Dudli S, Slaby O
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Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes

Previously, we proposed the hypothesis that similarities in the inflammatory response observed in acne vulgaris and degenerative disc disease (DDD), especially the central role of interleukin (IL)-1 beta, may be further evidence of the role of the anaerobic bacterium Cutibacterium (previously Propionibacterium) acnes in the underlying aetiology of disc degeneration. To investigate this, we examined the upregulation of IL-1 beta, and other known IL-1 beta-induced inflammatory markers and neurotrophic factors, from nucleus-pulposus-derived disc cells infected in vitro with C. acnes for up to 48 h. Upon infection, significant upregulation of IL-1 beta, alongside IL-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was observed with cells isolated from the degenerative discs of eight patients versus non-infected controls. Expression levels did, however, depend on gene target, multiplicity and period of infection and, notably, donor response. Pre-treatment of cells with clindamycin prior to infection significantly reduced the production of pro-inflammatory mediators. This study confirms that C. acnes can stimulate the expression of IL-1 beta and other host molecules previously associated with pathological changes in disc tissue, including neo-innervation. While still controversial, the role of C. acnes in DDD remains biologically credible, and its ability to cause disease likely reflects a combination of factors, particularly individualised response to infection.
Babino A, Magnasco MO
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Masks and distancing during COVID-19: a causal framework for imputing value to public-health interventions

SCIENTIFIC REPORTS 2021 MAR 4; 11(1):? Article 5183
During the COVID-19 pandemic, the scientific community developed predictive models to evaluate potential governmental interventions. However, the analysis of the effects these interventions had is less advanced. Here, we propose a data-driven framework to assess these effects retrospectively. We use a regularized regression to find a parsimonious model that fits the data with the least changes in the Rt parameter. Then, we postulate each jump in Rt as the effect of an intervention. Following the do-operator prescriptions, we simulate the counterfactual case by forcing Rt to stay at the pre-jump value. We then attribute a value to the intervention from the difference between true evolution and simulated counterfactual. We show that the recommendation to use facemasks for all activities would reduce the number of cases by 200,000 (95% CI 190,000-210,000) in Connecticut, Massachusetts, and New York State. The framework presented here might be used in any case where cause and effects are sparse in time.
Makwana K, Chodavarapu H, Morones N, Chi JY, Barr W, Novinbakht E, Wang YD, Nguyen PT, Jovanovic P, Cohen P, Riera CE
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Sensory neurons expressing calcitonin gene-related peptide a regulate adaptive thermogenesis and diet-induced obesity

MOLECULAR METABOLISM 2021 MAR; 45(?):? Article 101161
Objectives: Heat-sensory neurons from the dorsal root ganglia (DRG) play a pivotal role in detecting the cutaneous temperature and transmission of external signals to the brain, ensuring the maintenance of thermoregulation. However, whether these thermoreceptor neurons contribute to adaptive thermogenesis remains elusive. It is also unknown whether these neurons play a role in obesity and energy metabolism. Methods: We used genetic ablation of heat-sensing neurons expressing calcitonin gene-related peptide a (CGRPa) to assess whole-body energy expenditure, weight gain, glucose tolerance, and insulin sensitivity in normal chow and high-fat diet-fed mice. Ex vivo lipolysis and transcriptional characterization were combined with adipose tissue-clearing methods to visualize and probe the role of sensory nerves in adipose tissue. Adaptive thermogenesis was explored using infrared imaging of intrascapular brown adipose tissue (iBAT), tail, and core temperature upon various stimuli including diet, external temperature, and the cooling agent icilin. Results: In this report, we show that genetic ablation of heat-sensing CGRPa neurons promotes resistance to weight gain upon high-fat diet (HFD) feeding and increases energy expenditure in mice. Mechanistically, we found that loss of CGRPa-expressing sensory neurons was associated with reduced lipid deposition in adipose tissue, enhanced expression of fatty acid oxidation genes, higher ex vivo lipolysis in primary white adipocytes, and increased mitochondrial respiration from iBAT. Remarkably, mice lacking CGRPa sensory neurons manifested increased tail cutaneous vasoconstriction at room temperature. This exacerbated cold perception was not associated with reduced core temperature, suggesting that heat production and heat conservation mechanisms were engaged. Specific denervation of CGRPa neurons in intrascapular BAT did not contribute to the increased metabolic rate observed upon global sensory denervation. Conclusions: Taken together, these findings highlight an important role of cutaneous thermoreceptors in regulating energy metabolism by triggering counter-regulatory responses involving energy dissipation processes including lipid fuel utilization and cutaneous vasodilation. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (
Schafer A, Muecksch F, Lorenzi JCC, Leist SR, Cipolla M, Bournazos S, Schmidt F, Maison RM, Gazumyan A, Martinez DR, Baric RS, Robbiani DF, Hatziioannou T, Ravetch JV, Bieniasz PD, Bowen RA, Nussenzweig MC, Sheahan TP
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Antibody potency, effector function, and combinations in protection and therapy for SARS-CoV-2 infection in vivo

JOURNAL OF EXPERIMENTAL MEDICINE 2021 MAR 1; 218(3):? Article e20201993
SARS-CoV-2, the causative agent of COVID-19, has been responsible for over 42 million infections and 1 million deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here, we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a Syrian hamster model of SARS-CoV-2 and in a mouse-adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). Antibody combinations were effective for prevention and in therapy when administered early. However, in vitro antibody neutralization potency did not uniformly correlate with in vivo protection, and some hu-mAbs were more protective in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors contributes to optimal protection against SARS-CoV-2 MA. The data indicate that intact effector function can affect hu-mAb protective activity and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.
Alvim R, Nagar K, Das S, Lebdai S, Wong N, Somma A, Hughes C, Thomas J, Monette S, Scherz A, Kim K, Grimm J, Coleman JA
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Positron Emission Tomography/Computed Tomography with Gallium-68-labeled Prostate-specific Membrane Antigen Detects Relapse After Vascular-targeted Photodynamic Therapy in a Prostate Cancer Model

Background: Evaluating the efficacy of focal therapy for prostate cancer is limited by current approaches and may be improved with biological imaging techniques. Objective: We assessed whether positron emission tomography/computed tomography with gallium-68-labeled prostate-specific membrane antigen (Ga-68-PSMA PET/CT) can be used to predict relapse after vascular-targeted photodynamic therapy (VTP). Design, setting, and participants: A total of 1 x 10(6) LNCaP cells were grafted subcutaneously in the flanks of 6-8-wk-old SCID mice. Of 24 mice with measurable tumors 6 wk after tumor implantation, 20 were treated with VTP (150 mW/cm(2)) to ablate the tumors. Blood prostate-specific antigen (PSA) levels were assessed, and Ga-68-PSMA PET/CT images were performed 1 d before VTP and 1 and 4 wk after. Outcome measurements and statistical analysis: Local tumor relapse was evaluated by histology, and tumors were analyzed by prostate-specific membrane antigen (PSMA) and PSA immunohistochemistry. T tests and Kruskal-Wallis tests were used to determine significance. Results and limitations: Four weeks after VTP, 11 (65%) mice had complete responses and six (35%) had tumor relapses confirmed by histology (hematoxylin and eosin, and PSMA immunohistochemistry). All mice with local relapse had positive Ga-68-PSMA PET/CT findings 4 wk after VTP; all complete responders did not. One week after VTP, the relapse detection sensitivity of Ga-68-PSMA PET/CT was 75%, whereas the sensitivity of PSA was only 33%. Compared with controls, relapsed tumors had a three-fold reduction in the number of cells with strong PSA staining by immunohistochemistry (1.5% vs 4.5%; p = 0.01). Conclusions: In a preclinical prostate cancer model, we show that Ga-68-PSMA PET/CT can identify and predict relapse earlier than blood PSA level. These findings support further testing in clinical trials. Patient summary: Positron emission tomography/computed tomography with gallium-68-labeled prostate-specific membrane antigen may be used to follow and evaluate treatment outcomes in men who receive focal therapy for prostate cancer. (C) 2019 Published by Elsevier B.V. on behalf of European Association of Urology.