Biology subjects

Young, D.

Publications and source records attributed to Young, D..

28 records · Page 2Linked to original sources

bioRxiv2022

Segmental flexibility of bispecific T-cell engagers regulates the dynamics of immune synapse formation

Bispecific T-cell engagers (TcEs) link T cell receptors to tumor-associated antigens on cancer cells, forming cytotoxic immunological synapses (IS). Close membrane-to-membrane contact ([≤]13 nm) has been proposed as a key mechanism of TcE function. To investigate this and identify potential additional mechanisms, we compared four immunoglobulin G1-based (IgG1) TcE Formats (A-D) targeting CD3{varepsilon} and Her2, designed to create varying intermembrane distances (A B=C>D. In a minimal system for IS formation on SLBs, TcE performance followed the trend A=B=C>D. Addition of close-contact requiring CD58 co-stimulation revealed phospholipase C-{gamma} activation matching cytotoxicity with A>B=C>D. Our findings suggest that, when adhesion is equivalent, TcE potency is determined by two parameters: contact distance and flexibility. Both the close/far-contact formation axis and the low/high flexibility axis significantly impact TcE potency, explaining the similar potency of Format B (close-contact/high flexibility) and C (far-contact/low flexibility). Significance statementBispecific T-cell engagers (TcEs) are immunotherapeutic drugs that trigger the destruction of cancer cells by linking T cells to cancer cell through specific surface molecules (antigens). We designed a series of TcEs with varying distances between their binding sites and flexibilities of the TcE-antigen complexes. By combining structural and functional analyses, we confirmed close-contact formation between T cells and cancer cells as a critical determinant, mediated by co-activating receptors. Furthermore, we also identified molecular flexibility of the TcE-antigen complex as a further critical parameter for TcE potency. These findings provide novel insights into TcE function and highlight the importance of both parameters for future research and the design of improved immunotherapies.

immunology↗

bioRxiv2021

Intranasal administration of a monoclonal neutralizing antibody protects mice against SARS-CoV-2 infection

Despite recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both, prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2 and retains activity against the variants of concern B.1.1.7 and B.1.351. Importantly, not only systemic but also intranasal application of DZIF-10c abolished presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies. Significance StatementMonoclonal neutralizing antibodies are important in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection. However, their intravenous application might lead to suboptimal bioavailability in the lung. We here precisely characterize a new monoclonal neutralizing antibody (DZIF-10c) that binds to the receptor binding domain of the spike protein of SARS-CoV-2. DZIF-10c neutralizes SARS-CoV-2 with exceptionally high potency and maintains activity against circulating variants of concern. The antibody has a favorable pharmacokinetic profile and protects mice from SARS-CoV-2 infection. Importantly, we show that intranasal administration of DZIF-10c generates protective efficacy. These results not only identify DZIF-10c as a novel highly potent neutralizing antibody, but further pave the way for a topical application of anti-SARS-CoV-2 antibodies.

microbiology↗

bioRxiv2020

Lymph flow directs rapid neutrophil positioning in the lymph node in infection

Soon after Staphylococcus aureus (S. aureus) skin infection, neutrophils infiltrate the LN via the high endothelial venules (HEVs) to restrain and kill the invading microbes to prevent systemic spread of microbes. In this study, we found that rapid neutrophil migration depends on lymph flow, through which inflammatory chemokines/cytokines produced in the infected tissue are transported to the LN. Without lymph flow, bacteria accumulation in the LN was insufficient to stimulate chemokine production or neutrophil migration. Oxazolone (OX)-induced skin inflammation impaired lymphatic function, and reduced chemokines in the LN after a secondary infection with S. aureus. Due to LN reconstruction and impaired conduit-mediated lymph flow, neutrophil preferentially transmigrated in HEVs located in the medullary sinus, where the HEVs remained exposed to lymph-borne chemokines. Altered neutrophil migration resulted in persistent infection in the LN. Our studies showed that lymph flow directed chemokine dispersal in the LN and ensured rapid neutrophil migration for timely immune protection in infection. The impaired lymph flow and neutrophil migration may contribute to the frequent infection in skin inflammation, such as atopic dermatitis.

immunology↗

bioRxiv2020

Charge Interactions in a Highly Charge-depleted Protein

Interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions. SignificanceElectrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.

biochemistry↗

bioRxiv2020

Redox response of iron-sulfur glutaredoxin GRXS17 activates its holdase activity to protect plants from heat stress

Living organisms use a large panel of mechanisms to protect themselves from environmental stress. Particularly, heat stress induces misfolding and aggregation of proteins which are guarded by chaperone systems. Here, we examine the function the glutaredoxin GRXS17, a member of thiol reductases families in the model plant Arabidopsis thaliana. GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin (TRX)-domain and three CGFS-active site motif-containing GRX-domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione (GSH)-dependent manner. As a Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, we showed that GRXS17 presents both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induces loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active site cysteines in the corresponding TRX domains. This oxidation leads to a shift of GRXS17 to a high-MW complex and thus, activates its holdase activity. Moreover, we demonstrate that GRXS17 is specifically involved in plant tolerance to moderate high temperature and protects root meristematic cells from heat-induced cell death. Finally, we showed that upon heat stress, GRXS17 changes its client proteins, possibly to protect them from heat injuries. Therefore, we propose that the iron-sulfur cluster enzyme glutaredoxin GRXS17 is an essential guard to protect proteins against moderate heat stress, likely through a redox-dependent chaperone activity. All in all, we reveal the mechanism of an Fe-S cluster-dependent activity shift, turning the holoenzyme GRXS17 into a holdase that prevents damage caused by heat stress.

plant biology↗

bioRxiv2019

Astrocyte-selective AAV-ADAMTS4 gene therapy combined with hindlimb rehabilitation promotes functional recovery after spinal cord injury

Chondroitin sulphate proteoglycans (CSPGs) are inhibitors to axon regeneration and plasticity. Bacterial chondroitinase ABC degrades CSPGs and has been extensively reported to be therapeutic after SCI but there remain concerns for its clinical translation. A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) is a human enzyme that catalyses the proteolysis of CSPG protein cores. Infusion of ADAMTS4 into the damaged spinal cord was previously shown to improve functional recovery after SCI, however, this therapy is limited in its enzyme form. Adeno-associated viral (AAV) vector gene therapy has emerged as the vector of choice for safe, robust and long-term transgene expression in the central nervous system. Here, an AAV expression cassette containing ADAMTS4 under the control of the astrocytic GfaABC1D promoter was packaged into an AAV5 vector. Sustained expression of ADAMTS4 was achieved in vitro and in vivo, leading to widespread degradation of CSPGs. AAV-ADAMTS4 resulted in significantly decreased lesion size, increased sprouting of hindlimb corticospinal tract axons, increased serotonergic fiber density caudal to the injury, and improved functional recovery after moderate contusive SCI. Hindlimb-specific exercise rehabilitation was used to drive neuroplasticity towards improving functional connections. The combination of hindlimb rehabilitation with AAV-ADAMTS4 led to enhanced functional recovery after SCI. Thus, widespread and long-term degradation of CSPGs through AAV-ADAMTS4 gene therapy in a combinational approach with rehabilitation represents a promising candidate for further preclinical development.

neuroscience↗

bioRxiv2019

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases have arisen as possible genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. Here, we expanded luciferase multiplexing in post-lysis endpoint luciferase assays from two towards six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. Using synthetic assembly cloning, all six luciferase reporter units are stitched together into one plasmid; facilitating delivery of all reporter units through a process we named solotransfection, minimizing experimental errors. We engineered a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We were able to monitor the effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, as well as its broad application across different research fields.

molecular biology↗

bioRxiv2019

Quantifying the roles of vomiting, diarrhea, and residents vs. staff in norovirus transmission in U.S. nursing home outbreaks

The role of individual case characteristics, such as symptoms or demographics, in norovirus transmissibility is poorly understood. Six nursing home norovirus outbreaks occurring in South Carolina, U.S. from 2014 to 2016 were examined. We aimed to quantify the contribution of symptoms and other case characteristics in norovirus transmission using the reproduction number (REi) as an estimate of individual case infectivity and to examine how transmission changes over the course of an outbreak. Individual estimates of REi were calculated using a maximum likelihood procedure to infer the average number of secondary cases generated by each case. The associations between case characteristics and REi were estimated using a multivariate mixed linear model. Outbreaks began with one to three index case(s) with large estimated REis (range: 1.48 to 8.70) relative to subsequent cases. Of the 209 cases, 155 (75%) vomited, 164 (79%) had diarrhea, and 158 (76%) were nursing home residents (vs. staff). Cases who vomited infected 2.74 (95% CI: 1.90, 3.94) more individuals than non-vomiters, cases with diarrhea infected 1.62 (95% CI: 1.09, 2.41) more individuals than cases without diarrhea, and resident-cases infected 1.69 (95% CI: 1.18, 2.42) more individuals than staff-cases. Index cases tended to be residents (vs. staff) who vomited and infected considerably more secondary cases compared to non-index cases. Results suggest that individuals, particularly residents, who vomit are more infectious and tend to drive norovirus transmission in U.S. nursing home norovirus outbreaks. While diarrhea also plays a role in norovirus transmission, it is to a lesser degree than vomiting in these settings. Results lend support for prevention and control measures that focus on cases who vomit, particularly if those cases are residents.\n\nAuthor summaryThe majority of all norovirus outbreaks reported to the CDC occur in long-term care facilities (LTCFs), including nursing homes, where older residents are at risk for more severe or prolonged infection. Because there is currently no publicly available norovirus vaccine, sound control measures are key to controlling norovirus outbreaks, but there is little evidence that standard control measures are effective in reducing the size and/or duration of LTCF norovirus outbreaks. Hence, studies leading to a better understanding of disease spread and prevention of additional cases, and thus more effective control measures, are needed. To this end, we aimed to quantify factors associated with norovirus transmission and to examine how transmission changes over the course of an outbreak. We show that vomiting and, to a lesser extent, diarrhea are critical in initiating and sustaining norovirus transmission in U.S. nursing home norovirus outbreaks. We also show that nursing home residents, rather than staff, are the primary drivers of transmission. Results suggest that control measures focusing on cases who vomit, particularly if those cases are residents, would be most effective at curtailing norovirus transmission in these settings.

epidemiology↗

bioRxiv2018

Dual RNAseq of human leprosy lesions identifies bacterial determinants linked to host immune response

To understand how the interaction between an intracellular bacterium and the host immune system contributes to outcome at the site of infection, we studied leprosy, a disease that forms a clinical spectrum, in which progressive infection by the intracellular bacterium Mycobacterium leprae is characterized by the production of type | IFNs and antibody production. We performed dual RNAseq on patient lesions, identifying a continuum of distinct bacterial states that are linked to the host immune response. The bacterial burden, represented by the fraction of bacterial transcripts, correlates with a host type | IFN gene signature, known to inhibit antimicrobial responses. Second, the bacterial transcriptional activity, defined by the bacterial mRNA/rRNA ratio, links bacterial heat shock proteins with the BAFF-BCMA host antibody response pathway. Our findings provide a platform for interrogation of host and pathogen transcriptomes at the site of infection, allowing insight into mechanisms of inflammation in human disease.

microbiology↗

bioRxiv2018

Genomic determinants of sympatric speciation of the Mycobacterium tuberculosis complex across evolutionary timescales.

BACKGROUNDModels on how bacterial lineages differentiate increase our understanding on early bacterial speciation events and about the genetic loci involved. Here, we analyze the population genomics events leading to the emergence of the tuberculosis pathogen.\n\nRESULTSThe emergence is characterized by a combination of recombination events involving core pathogenesis functions and purifying selection on early diverging loci. We identify the phoR gene, the sensor kinase of a two-component system involved in virulence, as a key functional player subject to pervasive positive selection after the divergence of the MTBC from its ancestor. Previous evidence showed that phoR mutations played a central role in the adaptation of the pathogen to different host species. Now we show that phoR have been under selection during the early spread of human tuberculosis, during later expansions and in on-going transmission events.\n\nCONCLUSIONSOur results show that linking pathogen evolution across evolutionary and epidemiological timescales point to past and present virulence determinants.

evolutionary biology↗