UMass Chan Faculty and Researcher Publications

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This collection showcases journal articles, preprints, book chapters, and other publications and presentations produced by faculty, postdocs, and researchers at UMass Chan Medical School.

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Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing
(2023-05-24) Yip, Mitchell; Chen, Jing; Zhi, Yan; Tran, Ngoc Tam; Namkung, Suk; Pastor, Eric; Gao, Guangping; Tai, Phillip W L; Horae Gene Therapy Center; Microbiology; Li Weibo Institute for Rare Diseases Research
Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the - genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the / plasmid occurs under several events and rcAAVs spawn from diverse clones.
Development of Respiratory Syncytial Virus Vaccine Candidates for the Elderly
(2023-05-31) Blanco, Jorge C G; Cullen, Lori M; Kamali, Arash; Sylla, Fatouomata Y D; Boukhvalova, Marina S; Morrison, Trudy G; Microbiology
Respiratory syncytial virus (RSV) is a significant threat to elderly populations and repeated infections that occur throughout life are poorly protective. To assess the role of prior RSV infections as well as elderly immune senescence on vaccine efficacy, we compared immune responses after virus-like particle (VLP) immunization of elderly cotton rats and young cotton rats, both previously RSV infected, in order to mimic the human population. We show that immunization of RSV-experienced young or elderly animals resulted in the same levels of anti-pre-F IgG, anti-G IgG, neutralizing antibody titers, and protection from challenge indicating that the delivery of F and G proteins in a VLP is equally effective in activation of protective responses in both elderly and young populations. Our results suggest that F and G protein-containing VLPs induce anti-RSV memory established in prior RSV infections equally well in both young and elderly animals and thus can be an effective vaccine for the elderly.
ATAXIA TELANGIECTASIA MUTATED PROTECTS AGAINST LIPOPOLYSACCARIDE-INDUCED BLOOD-BRAIN BARRIER DISRUPTION BY REGULATING ATK/DRP1-MEDIATED MITOCHONDRIAL HOMEOSTASIS
(2023-05-05) Luo, Shiyuan; Lyu, Zhuochen; Ge, Lingling; Li, Yinjiao; Liu, Yuqi; Yuan, Yuan; Zhao, Rui; Huang, Lei; Zhao, Jianyuan; Huang, Hongjun; Luo, Yan; Molecular, Cell and Cancer Biology; Program in Molecular Medicine
Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of ATM in lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) disruption remains unknown. This study aimed to investigate the role and underlying mechanism of ATM in the regulation of the BBB function in sepsis. Methods: We used LPS to induce BBB disruption in vivo and to establish an in vitro model of cerebrovascular endothelial cells. Blood-brain barrier disruption was assessed by measuring Evans blue leakage and expression of vascular permeability regulators. To investigate the role of ATM, its inhibitor AZD1390 and clinically approved doxorubicin, an anthracycline that can activate ATM, were administered as scheduled. To explore the underlying mechanism, protein kinase B (AKT) inhibitor MK-2206 was administered to block the AKT/dynamin-related protein 1 (DRP1) pathway. Results: Lipopolysaccharide challenge induced significant BBB disruption, ATM activation, and mitochondrial translocation. Inhibiting ATM with AZD1390 aggravated BBB permeability as well as the following neuroinflammation and neuronal injury, while activation of ATM by doxorubicin abrogated these defects. Further results obtained in brain microvascular endothelial cells showed that ATM inhibition reduced the phosphorylation of DRP1 at serine (S) 637, promoted excessive mitochondrial fission, and resulted in mitochondrial malfunction. By activating ATM, doxorubicin increased the protein binding between ATM and AKT and promoted the phosphorylated activation of AKT at S473, which could directly phosphorylate DRP1 at S637 to repress excessive mitochondrial fission. Consistently, the protective role of ATM was abolished by the AKT inhibitor MK-2206. Conclusions: Ataxia telangiectasia mutated protects against LPS-induced BBB disruption by regulating mitochondrial homeostasis, at least in part, through the AKT/DRP1 pathway.
Inhibition of VEGF binding to neuropilin-2 enhances chemosensitivity and inhibits metastasis in triple-negative breast cancer
(2023-05-03) Xu, Zhiwen; Goel, Hira Lal; Burkart, Christoph; Burman, Luke; Chong, Yeeting E; Barber, Alison G; Geng, Yanyan; Zhai, Liting; Wang, Mengdie; Kumar, Ayush; Menefee, Ann; Polizzi, Clara; Eide, Lisa; Rauch, Kaitlyn; Rahman, Justin; Hamel, Kristina; Fogassy, Zachary; Klopp-Savino, Sofia; Paz, Suzanne; Zhang, Mingjie; Cubitt, Andrea; Nangle, Leslie A; Mercurio, Arthur M; Molecular, Cell and Cancer Biology
Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.
Plasmodium falciparum gametocyte carriage in longitudinally monitored incident infections is associated with duration of infection and human host factors
(2023-05-01) Andolina, Chiara; Ramjith, Jordache; Rek, John; Lanke, Kjerstin; Okoth, Joseph; Grignard, Lynn; Arinaitwe, Emmanuel; Briggs, Jessica; Bailey, Jeffrey; Aydemir, Ozkan; Kamya, Moses R; Greenhouse, Bryan; Dorsey, Grant; Staedke, Sarah G; Drakeley, Chris; Jonker, Marianne; Bousema, Teun; Program in Molecular Medicine
Malaria transmission depends on the presence of Plasmodium gametocytes that are the only parasite life stage that can infect mosquitoes. Gametocyte production varies between infections and over the course of infections. Infection duration is highly important for gametocyte production but poorly quantified. Between 2017 and 2019 an all-age cohort of individuals from Tororo, eastern Uganda was followed by continuous passive and routine assessments. We longitudinally monitored 104 incident infections from 98 individuals who were sampled once every 28 days and on any day of symptoms. Among infections that lasted ≥ 3 months, gametocyte appearance was near-universal with 96% of infections having detectable gametocytes prior to clearance. However, most infections were of much shorter duration; 55.7% of asymptomatic infections were detected only once. When considering all asymptomatic infections, regardless of their duration, only 36.3% had detectable gametocytes on at least one time-point prior to parasite clearance. Infections in individuals with sickle-cell trait (HbAS) were more likely to have gametocytes detected (Hazard Rate (HR) = 2.68, 95% CI 1.12, 6.38; p = 0.0231) and had gametocytes detected at higher densities (Density Ratio (DR) = 9.19, 95% CI 2.79, 30.23; p = 0.0002) compared to infections in wildtype (HbAA) individuals. Our findings suggest that a large proportion of incident infections is too short in duration and of too low density to contribute to onward transmission.
A multi-center, prospective cohort study of whole blood gene expression in the tuberculosis-diabetes interaction
(2023-05-12) Queiroz, Artur T L; Vinhaes, Caian L; Fukutani, Eduardo R; Gupte, Akshay N; Kumar, Nathella Pavan; Fukutani, Kiyoshi F; Arriaga, María B; Sterling, Timothy R; Babu, Subash; Gaikwad, Sanjay; Karyakarte, Rajesh; Mave, Vidya; Paradhkar, Mandar; Viswanathan, Vijay; Gupta, Amita; Andrade, Bruno B; Kornfeld, Hardy; Medicine
Diabetes mellitus (DM) increases tuberculosis (TB) severity. We compared blood gene expression in adults with pulmonary TB, with or without diabetes mellitus (DM) from sites in Brazil and India. RNA sequencing (RNAseq) performed at baseline and during TB treatment. Publicly available baseline RNAseq data from South Africa and Romania reported by the TANDEM Consortium were also analyzed. Across the sites, differentially expressed genes varied for each condition (DM, TB, and TBDM) and no pattern classified any one group across all sites. A concise signature of TB disease was identified but this was expressed equally in TB and TBDM. Pathway enrichment analysis failed to distinguish TB from TBDM, although there was a trend for greater neutrophil and innate immune pathway activation in TBDM participants. Pathways associated with insulin resistance, metabolic dysfunction, diabetic complications, and chromosomal instability were positively correlated with glycohemoglobin. The immune response to pulmonary TB as reflected by whole blood gene expression is substantially similar with or without comorbid DM. Gene expression pathways associated with the microvascular and macrovascular complications of DM are upregulated during TB, supporting a syndemic interaction between these coprevalent diseases.
Convergent genomic signatures of local adaptation across a continental-scale environmental gradient
(2023-05-19) Moreira, Lucas R; Smith, Brian Tilston; Program in Bioinformatics and Integrative Biology
Convergent local adaptation offers a glimpse into the role of constraint and stochasticity in adaptive evolution, in particular the extent to which similar genetic mechanisms drive adaptation to common selective forces. Here, we investigated the genomics of local adaptation in two nonsister woodpeckers that are codistributed across an entire continent and exhibit remarkably convergent patterns of geographic variation. We sequenced the genomes of 140 individuals of Downy () and Hairy () woodpeckers and used a suite of genomic approaches to identify loci under selection. We showed evidence that convergent genes have been targeted by selection in response to shared environmental pressures, such as temperature and precipitation. Among candidates, we found multiple genes putatively linked to key phenotypic adaptations to climate, including differences in body size (e.g., ) and plumage (e.g., ). These results are consistent with genetic constraints limiting the pathways of adaptation to broad climatic gradients, even after genetic backgrounds diverge.
Rit2 silencing in dopamine neurons drives a progressive Parkinsonian phenotype [preprint]
(2023-05-25) Kearney, Patrick J; Zhang, Yuanxi; Tan, Yanglan; Kahuno, Elizabeth; Conklin, Tucker L; Fagan, Rita R; Pavchinskiy, Rebecca G; Shafer, Scott A; Yue, Zhenyu; Melikian, Haley E; Brudnick Neuropsychiatric Research Institute; Neurobiology; Morningside Graduate School of Biomedical Sciences; Biochemistry and Molecular Biotechnology
Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression anomalies to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove a progressive motor dysfunction that was more rapid in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreases in DA release, striatal DA content, phenotypic DAergic markers, and a loss of DA neurons, with increased pSer129-alpha synuclein expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.
oskar acts with the transcription factor Creb to regulate long-term memory in crickets
(2023-05-16) Kulkarni, Arpita; Ewen-Campen, Ben; Terao, Kanta; Matsumoto, Yukihisa; Li, Yaolong; Watanabe, Takayuki; Kao, Jonchee A; Parhad, Swapnil S; Ylla, Guillem; Mizunami, Makoto; Extavour, Cassandra G; Program in Molecular Medicine
Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene was first identified based on its role in establishing the germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for . We show that is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket . In these stem cells, called neuroblasts, is required together with the ancient animal transcription factor to regulate long-term (but not short-term) olfactory memory. We provide evidence that positively regulates , which plays a conserved role in long-term memory across animals, and that in turn may be a direct target of Creb. Together with previous reports of a role for in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that 's original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene in the nervous system may have facilitated 's later co-option to the germ line in holometabolous insects.
Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis
(2023-05-04) Stavre, Zheni; Kim, Jung-Min; Yang, Yeon-Suk; Nündel, Kerstin; Chaugule, Sachin; Sato, Tadatoshi; Park, Kwang Hwan; Gao, Guangping; Gravallese, Ellen M; Shim, Jae-Hyuck; Medicine; Horae Gene Therapy Center; Li Weibo Institute for Rare Diseases Research; Microbiology
Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/β-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/β-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA.
One Step Purification-Vaccine Delivery System
(2023-05-01) Soto, Ernesto R; Specht, Charles A; Lee, Chrono K; Levitz, Stuart M; Ostroff, Gary R; Program in Molecular Medicine; Medicine
Glucan particles (GPs) are hollow, porous 3-5 µm microspheres derived from the cell walls of Baker's yeast (). Their 1,3-β-glucan outer shell allows for receptor-mediated uptake by macrophages and other phagocytic innate immune cells expressing β-glucan receptors. GPs have been used for the targeted delivery of a wide range of payloads, including vaccines and nanoparticles, encapsulated inside the hollow cavity of GPs. In this paper, we describe the methods to prepare GP-encapsulated nickel nanoparticles (GP-Ni) for the binding of histidine (His)-tagged proteins. His-tagged Cda2 cryptococcal antigens were used as payloads to demonstrate the efficacy of this new GP vaccine encapsulation approach. The GP-Ni-Cda2 vaccine was shown to be comparable to our previous approach utilizing mouse serum albumin (MSA) and yeast RNA trapping of Cda2 in GPs in a mouse infection model. This novel GP-Ni approach allows for the one-step binding of His-tagged vaccine antigens and encapsulation in an effective delivery vehicle to target vaccines to antigen-presenting cells (APCs), antigen discovery, and vaccine development.
Assessing pain catastrophizing and functional disability in pediatric epidermolysis bullosa patients
(2022-12-29) Rangu, Sneha; Collins, Jessica; García-Romero, Maria Teresa; Augsburger, Bret D; Bruckner, Anna L; Diaz, Lucia Z; Eichenfield, Lawrence F; Faig, Walter; Gorell, Emily S; Lefferdink, Rachel; Lucky, Anne W; Morel, Kimberly D; Paller, Amy S; Park, Helen; Pastrana-Arellano, Elena; Peoples, Kathleen; Wiss, Karen; Perman, Marissa J; Castelo-Soccio, Leslie; Dermatology; Pediatrics
Background/objectives: The primary objective was to assess pain catastrophizing and functional disability in pediatric patients with epidermolysis bullosa (EB) and their parents/guardians. Secondary objectives included examining relationships between pain catastrophizing, functional disability, and correlations with other factors (e.g., age, disease severity, and percent of body surface area (BSA) involved). Methods: Patients with EB ages 8-16 and their parents/guardians who were English or Spanish speaking completed a one-time online survey. Parent measures included: demographics questionnaire, Pain Catastrophizing Scale-Parent (PCS), and Parent Functional Disability Inventory (FDI). Child measures included: PCS child and child FDI. Higher scores on both scales indicate higher levels of catastrophizing and functional disability. Results: Of 31 children, the mean age was 11.47 years and the majority (70.97%) had dystrophic EB. Mean scores were: 35.84 = PCS parent; 34.58 = PCS child; 30.87 = parent FDI; 29.77 = child FDI. Total scores for PCS parent, parent FDI, and child FDI increased significantly with disease severity and percentage of involved BSA (p < .01 for all). Total scores for PCS child increased significantly with percent of EB skin involvement (p = .04) but not disease severity. Older children reported more functional disability than their parents and younger children (p = .02). Conclusions: Our results demonstrate significant positive correlations between negative thoughts related to pain and the experience of functional difficulties in patients with EB and their caregivers. Psychological, psychiatric, and/or behavioral interventions to help managing chronic pain may be effective for patients with EB.
ERα-associated translocations underlie oncogene amplifications in breast cancer
(2023-05-17) Lee, Jake June-Koo; Jung, Youngsook Lucy; Cheong, Taek-Chin; Espejo Valle-Inclan, Jose; Chu, Chong; Gulhan, Doga C; Ljungström, Viktor; Jin, Hu; Viswanadham, Vinayak V; Watson, Emma V; Cortés-Ciriano, Isidro; Elledge, Stephen J; Chiarle, Roberto; Pellman, David; Park, Peter J; Systems Biology
Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure and the evolutionary trajectories of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.
ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain
(2023-05-11) Stratoulias, Vassilis; Ruiz, Rocío; Kanatani, Shigeaki; Osman, Ahmed M; Keane, Lily; Armengol, Jose A; Rodríguez-Moreno, Antonio; Murgoci, Adriana-Natalia; García-Domínguez, Irene; Alonso-Bellido, Isabel; González Ibáñez, Fernando; Picard, Katherine; Vázquez-Cabrera, Guillermo; Posada-Pérez, Mercedes; Vernoux, Nathalie; Tejera, Dario; Grabert, Kathleen; Cheray, Mathilde; González-Rodríguez, Patricia; Pérez-Villegas, Eva M; Martínez-Gallego, Irene; Lastra-Romero, Alejandro; Brodin, David; Avila-Cariño, Javier; Cao, Yang; Airavaara, Mikko; Uhlén, Per; Heneka, Michael T; Tremblay, Marie-Ève; Blomgren, Klas; Venero, Jose L; Joseph, Bertrand; Medicine
Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1 microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1 microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1 microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.
Candida expansion in the gut of lung cancer patients associates with an ecological signature that supports growth under dysbiotic conditions
(2023-05-09) Seelbinder, Bastian; Lohinai, Zoltan; Vazquez-Uribe, Ruben; Brunke, Sascha; Chen, Xiuqiang; Mirhakkak, Mohammad; Lopez-Escalera, Silvia; Dome, Balazs; Megyesfalvi, Zsolt; Berta, Judit; Galffy, Gabriella; Dulka, Edit; Wellejus, Anja; Weiss, Glen J; Bauer, Michael; Hube, Bernhard; Sommer, Morten O A; Panagiotou, Gianni; Medicine
Candida species overgrowth in the human gut is considered a prerequisite for invasive candidiasis, but our understanding of gut bacteria promoting or restricting this overgrowth is still limited. By integrating cross-sectional mycobiome and shotgun metagenomics data from the stool of 75 male and female cancer patients at risk but without systemic candidiasis, bacterial communities in high Candida samples display higher metabolic flexibility yet lower contributional diversity than those in low Candida samples. We develop machine learning models that use only bacterial taxa or functional relative abundances to predict the levels of Candida genus and species in an external validation cohort with an AUC of 78.6-81.1%. We propose a mechanism for intestinal Candida overgrowth based on an increase in lactate-producing bacteria, which coincides with a decrease in bacteria that regulate short chain fatty acid and oxygen levels. Under these conditions, the ability of Candida to harness lactate as a nutrient source may enable Candida to outcompete other fungi in the gut.
Mechanism of action for small-molecule inhibitors of triacylglycerol synthesis
(2023-05-29) Sui, Xuewu; Wang, Kun; Song, Kangkang; Xu, Chen; Song, Jiunn; Lee, Chia-Wei; Liao, Maofu; Farese, Robert V; Walther, Tobias C; Biochemistry and Molecular Biotechnology
Inhibitors of triacylglycerol (TG) synthesis have been developed to treat metabolism-related diseases, but we know little about their mechanisms of action. Here, we report cryo-EM structures of the TG-synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a membrane bound O-acyltransferase (MBOAT), in complex with two different inhibitors, T863 and DGAT1IN1. Each inhibitor binds DGAT1's fatty acyl-CoA substrate binding tunnel that opens to the cytoplasmic side of the ER. T863 blocks access to the tunnel entrance, whereas DGAT1IN1 extends further into the enzyme, with an amide group interacting with more deeply buried catalytic residues. A survey of DGAT1 inhibitors revealed that this amide group may serve as a common pharmacophore for inhibition of MBOATs. The inhibitors were minimally active against the related MBOAT acyl-CoA:cholesterol acyltransferase 1 (ACAT1), yet a single-residue mutation sensitized ACAT1 for inhibition. Collectively, our studies provide a structural foundation for developing DGAT1 and other MBOAT inhibitors.
A2Ar-dependent PD-1+ and TIGIT+ Treg cells have distinct homing requirements to suppress autoimmune uveitis in mice
(2023-05-09) Peters, Kayleigh; McDonald, Trisha; Muhammad, Fauziyya; Walsh, Marisa; Drenen, Kayla; Montieth, Alyssa; Stephen Foster, C; Lee, Darren J; Ophthalmology and Visual Sciences
The proper function of regulatory T cells (Tregs) to suppress inflammation requires homing to the correct tissue site. Resolution of autoimmune uveitis generates distinct programmed death receptor 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) Tregs in an adenosine 2A receptor (A2Ar)-dependent manner found in the spleen. Where and how these Tregs migrate from the spleen to prevent uveitis is not known. In this work, we show that A2Ar-dependent Tregs migrated to the eye and secondary lymphoid tissue and expressed chemokine receptor (CCR)6 and CCR7. Suppression of autoimmune uveitis required CCR6 and CCR7 expression for TIGIT Tregs but not PD-1 Tregs. Moreover, stimulation of A2Ar on T cells from patients showed a decreased capacity to induce TIGIT Tregs that expressed CCR6 or CCR7, and PD-1 Treg that expressed CCR6. This work provides a mechanistic understanding of the homing requirements of each of these Treg populations. Importantly, this work is clinically relevant because patients with chronic autoimmune uveitis are unable to induce the Treg populations identified in mice that home to the target tissue.
Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate
(2023-05-09) Nandadasa, Sumeda; Martin, Daniel; Deshpande, Gauravi; Robert, Karyn L; Stack, M Sharon; Itoh, Yoshifumi; Apte, Suneel S; Pediatrics
The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr-Gly bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.
Susceptibility of Ugandan Plasmodium falciparum Isolates to the Antimalarial Drug Pipeline
(2023-05-09) Kreutzfeld, Oriana; Tumwebaze, Patrick K; Okitwi, Martin; Orena, Stephen; Byaruhanga, Oswald; Katairo, Thomas; Conrad, Melissa D; Rasmussen, Stephanie A; Legac, Jennifer; Aydemir, Ozkan; Giesbrecht, David; Forte, Barbara; Campbell, Peter; Smith, Alasdair; Kano, Hiroki; Nsobya, Samuel L; Blasco, Benjamin; Duffey, Maelle; Bailey, Jeffrey A; Cooper, Roland A; Rosenthal, Philip J; Program in Molecular Medicine
Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome , dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median ICs, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected with drug pressure, and none were associated with significantly decreased drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.
Utilization of whole health services among veterans with spinal cord injuries and disorders (SCI/D): Early insights from the VA SCI/D system of care
(2023-05-30) Wallen, Erik S; Sippel, Jennifer L; Park, Meaghan E; Etingen, Bella; Weaver, Frances M; Hogan, Timothy P; Smith, Bridget M; Bokhour, Barbara G; Wickremasinghe, Itala M; Population and Quantitative Health Sciences
Context/objective: Our objective was to describe early participation in Whole Health programs across the Veterans Health Administration (VHA) Spinal Cord Injuries and Disorders (SCI/D) System of Care. Design: Retrospective analysis of VHA administrative data. Setting: The VHA SCI/D System of Care. Participants: Veterans with SCI/D included in the FY2019 cumulative VHA SCI/D Registry cohort with living status during FY2017, FY2018, and FY2019. Interventions: N/A. Outcome measures: We assessed the number of encounters and unique Veterans with SCI/D, and the percent of Veterans with SCI/D, who utilized each Whole Health (WH) program available in VA. Results: Utilization of WH Pathway and well-being Programs increased from 62 encounters to 1703 encounters between FY2017 and FY2019 (representing 0.09% to 3.13% of Veterans with SCI/D). Utilization of chiropractic care rose from 130 encounters to 418 encounters during the same time period. Similarly, utilization of complementary and integrative health programs increased from 886 encounters to 2655 encounters (representing 1.09% to 3.11% of Veterans; FY2017 to 2019). We also report utilization of specific WH programs. Conclusion: Participation in WH services has been increasing among Veterans with SCI/D who receive health care from the VHA SCI/D System of Care. However, utilization among Veterans with SCI/D remains low overall, and targeted efforts to increase WH program reach are needed. Additional information about the relative effectiveness of different strategies to support WH implementation is also needed, to ensure strategies likely to have the most impact are prioritized.

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