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person:Karim-Jean Armache (armack01) or Joel Belasco (belasj01) or bhabhg01 or burdes01 or cadwek01 or chaom01 or ekierd01 or froemr01 or gelmaj01 or jah12 or hubbas01 or knauth01 or lafaij01 or littmd01 or nancej01 or narask01 or neubet01 or novicr01 or ringsn01 or schwas13 or sfeira01 or skolne01 or smiths04 or stoked01 or torrej12 or treisj01 or turnbd01 or wangd01 or rifkid01 or ryooh01 or wilsoe01



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Locus coeruleus activity improves cochlear implant performance

Glennon, Erin; Valtcheva, Silvana; Zhu, Angela; Wadghiri, Youssef Z; Svirsky, Mario A; Froemke, Robert C
Cochlear implants (CIs) are neuroprosthetic devices that can provide hearing to deaf people1. Despite the benefits offered by CIs, the time taken for hearing to be restored and perceptual accuracy after long-term CI use remain highly variable2,3. CI use is believed to require neuroplasticity in the central auditory system, and differential engagement of neuroplastic mechanisms might contribute to the variability in outcomes4-7. Despite extensive studies on how CIs activate the auditory system4,8-12, the understanding of CI-related neuroplasticity remains limited. One potent factor enabling plasticity is the neuromodulator noradrenaline from the brainstem locus coeruleus (LC). Here we examine behavioural responses and neural activity in LC and auditory cortex of deafened rats fitted with multi-channel CIs. The rats were trained on a reward-based auditory task, and showed considerable individual differences of learning rates and maximum performance. LC photometry predicted when CI subjects began responding to sounds and longer-term perceptual accuracy. Optogenetic LC stimulation produced faster learning and higher long-term accuracy. Auditory cortical responses to CI stimulation reflected behavioural performance, with enhanced responses to rewarded stimuli and decreased distinction between unrewarded stimuli. Adequate engagement of central neuromodulatory systems is thus a potential clinically relevant target for optimizing neuroprosthetic device use.
PMID: 36544024
ISSN: 1476-4687
CID: 5395022

Longitudinal in Utero Analysis of Engrailed-1 Knockout Mouse Embryonic Phenotypes Using High-Frequency Ultrasound

Aristizábal, Orlando; Qiu, Ziming; Gallego, Estefania; Aristizábal, Matias; Mamou, Jonathan; Wang, Yao; Ketterling, Jeffrey A; Turnbull, Daniel H
Large-scale international efforts to generate and analyze loss-of-function mutations in each of the approximately 20,000 protein-encoding gene mutations are ongoing using the "knockout" mouse as a model organism. Because one-third of gene knockouts are expected to result in embryonic lethality, it is important to develop non-invasive in utero imaging methods to detect and monitor mutant phenotypes in mouse embryos. We describe the utility of 3-D high-frequency (40-MHz) ultrasound (HFU) for longitudinal in utero imaging of mouse embryos between embryonic days (E) 11.5 and E14.5, which represent critical stages of brain and organ development. Engrailed-1 knockout (En1-ko) mouse embryos and their normal control littermates were imaged with HFU in 3-D, enabling visualization of morphological phenotypes in the developing brains, limbs and heads of the En1-ko embryos. Recently developed deep learning approaches were used to automatically segment the embryonic brain ventricles and bodies from the 3-D HFU images, allowing quantitative volumetric analyses of the En1-ko brain phenotypes. Taken together, these results show great promise for the application of longitudinal 3-D HFU to analyze knockout mouse embryos in utero.
PMID: 36283941
ISSN: 1879-291x
CID: 5359402

Stable Isotope Labeling by Amino Acids and Bioorthogonal Noncanonical Amino Acid Tagging in Cultured Primary Neurons

Zhang, Guoan; Deinhardt, Katrin; Neubert, Thomas A
Cultured primary neurons are a well-established model for the study of neuronal function. Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires nearly complete metabolic labeling of proteins and therefore is difficult to apply to cultured primary neurons, which do not divide in culture. In a multiplex SILAC strategy, two different sets of heavy amino acids are used for labeling cells for the different experimental conditions. This allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. When combined with bioorthogonal noncanonical amino acid tagging (BONCAT), it allows for comparative proteomic analysis of de novo protein synthesis. Here we describe protocols that utilize the multiplex SILAC labeling strategy for primary cultured neurons to study steady-state and nascent proteomes.
PMID: 36370278
ISSN: 1940-6029
CID: 5357722

Gut microbiome of helminth-infected indigenous Malaysians is context dependent

Tee, Mian Zi; Er, Yi Xian; Easton, Alice V; Yap, Nan Jiun; Lee, Ii Li; Devlin, Joseph; Chen, Ze; Ng, Kee Seong; Subramanian, Poorani; Angelova, Angelina; Oyesola, Oyebola; Sargsian, Shushan; Ngui, Romano; Beiting, Daniel P; Boey, Christopher Chiong Meng; Chua, Kek Heng; Cadwell, Ken; Lim, Yvonne Ai Lian; Loke, P'ng; Lee, Soo Ching
BACKGROUND:While microbiomes in industrialized societies are well characterized, indigenous populations with traditional lifestyles have microbiomes that are more akin to those of ancient humans. However, metagenomic data in these populations remains scarce, and the association with soil-transmitted helminth infection status is unclear. Here, we sequenced 650 metagenomes of indigenous Malaysians from five villages with different prevalence of helminth infections. RESULTS:Individuals from villages with higher prevalences of helminth infections have more unmapped reads and greater microbial diversity. Microbial community diversity and composition were most strongly associated with different villages and the effects of helminth infection status on the microbiome varies by village. Longitudinal changes in the microbiome in response to albendazole anthelmintic treatment were observed in both helminth infected and uninfected individuals. Inference of bacterial population replication rates from origin of replication analysis identified specific replicating taxa associated with helminth infection. CONCLUSIONS:Our results indicate that helminth effects on the microbiota were highly dependent on context, and effects of albendazole on the microbiota can be confounding for the interpretation of deworming studies. Furthermore, a substantial quantity of the microbiome remains unannotated, and this large dataset from an indigenous population associated with helminth infections is a valuable resource for future studies. Video Abstract.
PMID: 36476263
ISSN: 2049-2618
CID: 5378722

De novo design of obligate ABC-type heterotrimeric proteins

Bermeo, Sherry; Favor, Andrew; Chang, Ya-Ting; Norris, Andrew; Boyken, Scott E; Hsia, Yang; Haddox, Hugh K; Xu, Chunfu; Brunette, T J; Wysocki, Vicki H; Bhabha, Gira; Ekiert, Damian C; Baker, David
The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.
PMID: 36522429
ISSN: 1545-9985
CID: 5382412

ADP-ribosyltransferases, an update on function and nomenclature

Lüscher, Bernhard; Ahel, Ivan; Altmeyer, Matthias; Ashworth, Alan; Bai, Peter; Chang, Paul; Cohen, Michael; Corda, Daniela; Dantzer, Françoise; Daugherty, Matthew D; Dawson, Ted M; Dawson, Valina L; Deindl, Sebastian; Fehr, Anthony R; Feijs, Karla L H; Filippov, Dmitri V; Gagné, Jean-Philippe; Grimaldi, Giovanna; Guettler, Sebastian; Hoch, Nicolas C; Hottiger, Michael O; Korn, Patricia; Kraus, W Lee; Ladurner, Andreas; Lehtiö, Lari; Leung, Anthony K L; Lord, Christopher J; Mangerich, Aswin; Matic, Ivan; Matthews, Jason; Moldovan, George-Lucian; Moss, Joel; Natoli, Gioacchino; Nielsen, Michael L; Niepel, Mario; Nolte, Friedrich; Pascal, John; Paschal, Bryce M; PawÅ‚owski, Krzysztof; Poirier, Guy G; Smith, Susan; Timinszky, Gyula; Wang, Zhao-Qi; Yélamos, José; Yu, Xiaochun; Zaja, Roko; Ziegler, Mathias
ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.
PMID: 34323016
ISSN: 1742-4658
CID: 5010662

Clostridia isolated from helminth-colonized humans promote the life cycle of Trichuris species

Sargsian, Shushan; Chen, Ze; Lee, Soo Ching; Robertson, Amicha; Thur, Rafaela Saes; Sproch, Julia; Devlin, Joseph C; Tee, Mian Zi; Er, Yi Xian; Copin, Richard; Heguy, Adriana; Pironti, Alejandro; Torres, Victor J; Ruggles, Kelly V; Lim, Yvonne A L; Bethony, Jeffrey; Loke, P'ng; Cadwell, Ken
Soil-transmitted intestinal worms known as helminths colonize over 1.5 billion people worldwide. Although helminth colonization has been associated with altered composition of the gut microbiota, such as increases in Clostridia, individual species have not been isolated and characterized. Here, we isolate and sequence the genome of 13 Clostridia from the Orang Asli, an indigenous population in Malaysia with a high prevalence of helminth infections. Metagenomic analysis of 650 fecal samples from urban and rural Malaysians confirm the prevalence of species corresponding to these isolates and reveal a specific association between Peptostreptococcaceae family members and helminth colonization. Remarkably, Peptostreptococcaceae isolated from the Orang Asli display superior capacity to promote the life cycle of whipworm species, including hatching of eggs from Trichuris muris and Trichuris trichiura. These findings support a model in which helminths select for gut colonization of microbes that support their life cycle.
PMID: 36450245
ISSN: 2211-1247
CID: 5374022

Defensosomes: a new role for autophagy proteins in innate immune defense

Ching, Krystal L; Torres, Victor J; Cadwell, Ken
In recent years, the contribution of exosomes to immunity, inflammation and host-pathogen interaction have been appreciated. Exosomes are small secreted extracellular vesicles from endosomal origin that contain a myriad of cellular molecules (protein, nucleic acids), including surface receptors. We have reported a pathogen-induced and macroautophagy/autophagy-dependent class of exosomes coined as "defensosomes", which protect the host from membrane-targeting toxins. In a recent study, we found that defensosomes decorated with ACE2, the SARS-CoV-2 cellular receptor, are produced in the lungs of patients with COVID-19, and that increased concentration of ACE2-loaded defensosomes is associated with decreased hospitalization length. Mechanistically, SARS-CoV-2 induces the production of ACE2-coated defensosomes, a process requiring the autophagy machinery, which in turn binds and neutralizes the virus. We propose that defensosomes represent a new form of autophagy-mediated innate immunity that contributes to the host's armamentarium against pathogens.
PMID: 36409156
ISSN: 1554-8635
CID: 5371952

Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element

Hall, Jason A; Pokrovskii, Maria; Kroehling, Lina; Kim, Bo-Ram; Kim, Seung Yong; Wu, Lin; Lee, June-Yong; Littman, Dan R
T helper 17 (Th17) cells regulate mucosal barrier defenses but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been elucidated, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor RORγt is critical for Th17 cell differentiation; however, it is not clear whether the closely related RORα, which is co-expressed in Th17 cells, has a distinct role. Here, we demonstrated that although dispensable for Th17 cell differentiation, RORα was necessary for optimal Th17 responses in peripheral tissues. The absence of RORα in T cells led to reductions in both RORγt expression and effector function among Th17 cells. Cooperative binding of RORα and RORγt to a previously unidentified Rorc cis-regulatory element was essential for Th17 lineage maintenance in vivo. These data point to a non-redundant role of RORα in Th17 lineage maintenance via reinforcement of the RORγt transcriptional program.
PMID: 36243007
ISSN: 1097-4180
CID: 5358332

Gut microbiome dysbiosis in antibiotic-treated COVID-19 patients is associated with microbial translocation and bacteremia

Bernard-Raichon, Lucie; Venzon, Mericien; Klein, Jon; Axelrad, Jordan E; Zhang, Chenzhen; Sullivan, Alexis P; Hussey, Grant A; Casanovas-Massana, Arnau; Noval, Maria G; Valero-Jimenez, Ana M; Gago, Juan; Putzel, Gregory; Pironti, Alejandro; Wilder, Evan; Thorpe, Lorna E; Littman, Dan R; Dittmann, Meike; Stapleford, Kenneth A; Shopsin, Bo; Torres, Victor J; Ko, Albert I; Iwasaki, Akiko; Cadwell, Ken; Schluter, Jonas
Although microbial populations in the gut microbiome are associated with COVID-19 severity, a causal impact on patient health has not been established. Here we provide evidence that gut microbiome dysbiosis is associated with translocation of bacteria into the blood during COVID-19, causing life-threatening secondary infections. We first demonstrate SARS-CoV-2 infection induces gut microbiome dysbiosis in mice, which correlated with alterations to Paneth cells and goblet cells, and markers of barrier permeability. Samples collected from 96 COVID-19 patients at two different clinical sites also revealed substantial gut microbiome dysbiosis, including blooms of opportunistic pathogenic bacterial genera known to include antimicrobial-resistant species. Analysis of blood culture results testing for secondary microbial bloodstream infections with paired microbiome data indicates that bacteria may translocate from the gut into the systemic circulation of COVID-19 patients. These results are consistent with a direct role for gut microbiome dysbiosis in enabling dangerous secondary infections during COVID-19.
PMID: 36319618
ISSN: 2041-1723
CID: 5358262