Biology subjects

Reddy, R.

Publications and source records attributed to Reddy, R..

26 records · Page 2Linked to original sources

bioRxiv2021

Glutamate-Weighted Magnetic Resonance Imaging (GluCEST) Detects Effects of Transcranial Magnetic Stimulation to the Motor Cortex

Transcranial magnetic stimulation (TMS) is used in several FDA-approved treatments and, increasingly, to treat neurological disorders in off-label uses. However, the mechanism by which TMS causes physiological change is unclear, as are the origins of response variability in the general population. Ideally, objective in vivo biomarkers could shed light on these unknowns and eventually inform personalized interventions. Continuous theta burst stimulation (cTBS) is a form of TMS which has been observed to reduce motor evoked potentials (MEPs) for 60 minutes or longer post-stimulation, although the consistency of this effect and its mechanism continue to be under debate. Here, we use glutamate-weighted chemical exchange saturation transfer (gluCEST) magnetic resonance imaging (MRI) at ultra-high magnetic field (7T) to measure changes in glutamate concentration at the site of cTBS. We find that gluCEST signal in the ipsilateral hemisphere of the brain generally decreases in response to cTBS, whereas consistent changes were not detected in the contralateral or in subjects receiving a sham stimulation. One Sentence SummaryWe used glutamate-weighted Chemical Exchange Saturation Transfer (GluCEST) imaging to detect changes in glutamate contrast in the brains of young, healthy adults undergoing transcranial magnetic stimulation (TMS) to the motor cortex.

neuroscience↗

bioRxiv2021

Extracellular Vesicles Deliver Mitochondria and HSP27 Protein to Protect the Blood-Brain Barrier

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of endothelial cell-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous heat shock protein 27 (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, which subsequently colocalized with the mitochondrial network of the recipient primary human BECs. BECs treated with m/lEVs increased relative ATP levels and displayed superior mitochondrial function. Importantly, m/lEVs isolated from oligomycin (mitochondrial complex V inhibitor) or rotenone (mitochondrial complex I inhibitor)-exposed BECs (RTN-m/lEVs or OGM-m/lEVs) did not increase BECs ATP levels compared to naive m/lEVs. In contrast, RTN-sEV and OGM-sEV functionality in increasing cellular ATP levels was minimally impacted in comparison to naive sEVs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EV with cationic polymer poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two-punch approach to increase BEC metabolic function and tight junction integrity is a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/466491v5_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@dc5493org.highwire.dtl.DTLVardef@134ad7forg.highwire.dtl.DTLVardef@16a8929org.highwire.dtl.DTLVardef@15315bc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMedium-to-large extracellular vesicles (m/lEVs), not small EVs contain mitochondria C_LIO_LIm/lEVs increased ATP and mitochondrial function in brain endothelial cells (BECs) C_LIO_LIm/lEVs from oligomycin-exposed BECs did not increase recipient BEC ATP levels C_LIO_LIIntravenously injected m/lEVs reduced brain infarct sizes in a mouse stroke model C_LIO_LIEV/HSP27 mixtures reduced small and large dextran molecule permeability across BECs C_LI

bioengineering↗

bioRxiv2021

MXtrodes: MXene-infused bioelectronic interfaces for multiscale electrophysiology and stimulation

Soft bioelectronic interfaces for mapping and modulating excitable networks at high resolution and at large scale can enable paradigm-shifting diagnostics, monitoring, and treatment strategies. Yet, current technologies largely rely on materials and fabrication schemes that are expensive, do not scale, and critically limit the maximum attainable resolution and coverage. Solution processing is a cost-effective manufacturing alternative, but biocompatible conductive inks matching the performance of conventional metals are lacking. Here, we introduce MXtrodes, a novel class of soft, high-resolution, large-scale bioelectronic interfaces enabled by Ti3C2 MXene and scalable solution processing. We show that the electrochemical properties of MXtrodes exceed those of conventional materials, and do not require conductive gels when used in epidermal electronics. Furthermore, we validate MXtrodes in a number of applications ranging from mapping large scale neuromuscular networks in humans to delivering cortical microstimulation in small animal models. Finally, we demonstrate that MXtrodes are compatible with standard clinical neuroimaging modalities.

bioengineering↗

bioRxiv2021

DNA-Encoded Multivalent Display of Protein Tetramers on Phage: Synthesis and In Vivo Aplications

Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a [~]200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/432100v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18edf0org.highwire.dtl.DTLVardef@ad55c6org.highwire.dtl.DTLVardef@1da47d8org.highwire.dtl.DTLVardef@1a2ec32_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

Haloarchaea are highly resistant to oxidative stress, however, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volcanii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative "seed" region within the predicted stem loop of SHOxi. Malic enzyme is an enzyme of the tricarboxylic acid cycle that catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

microbiology↗

bioRxiv2020

Genetically Encoded, Multivalent Liquid Glycan Array (LiGA)

The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a "Liquid Glycan Array" (LiGA) platform comprising a library of DNA barcoded M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo; measurements that cannot be performed with canonical glass slide-based glycan arrays. DedicationThe paper is dedicated to Laura L. Kiessling on the occasion of her 60th birthday.

biochemistry↗

bioRxiv2020

Ribosome Profiling in Archaea Reveals Leaderless Translation, Novel Translational Initiation Sites, and Ribosome Pausing at Single Codon Resolution

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

molecular biology↗

bioRxiv2019

Regulatory non-coding small RNAs are diverse and abundant in an extremophilic microbial community

Regulatory small RNAs (sRNAs) represent a major class of regulatory molecules that play large-scale and essential roles in many cellular processes across all domains of life. Microbial sRNAs have been primarily investigated in a few model organisms and little is known about the dynamics of sRNA synthesis in natural environments, and the roles of these short transcripts at the community level. Analyzing the metatranscriptome of a model extremophilic community inhabiting halite nodules (salt rocks) from the Atacama Desert with SnapT - a new sRNA annotation pipeline - we discovered hundreds of intergenic (itsRNAs) and antisense (asRNAs) sRNAs. The halite sRNAs were taxonomically diverse with the majority expressed by members of the Halobacteria. We found asRNAs with expression levels negatively correlated with that of their putative overlapping target, suggesting a potential gene regulatory mechanism. A number of itsRNAs were conserved and significantly differentially expressed (FDR<5%) between 2 sampling time points allowing for stable secondary structure modeling and target prediction. This work demonstrates that metatranscriptomic field experiments link environmental variation with changes in RNA pools and have the potential to provide new insights into environmental sensing and responses in natural microbial communities through non-coding RNA mediated gene regulation.

microbiology↗