Biology subjects

Teng, B.

Publications and source records attributed to Teng, B..

7 recordsLinked to original sources

bioRxiv2025

Ultrahigh-Throughput Forster Resonance Energy Transfer-Activated Droplet Sorting for Terminator Polymerase Engineering

Droplet microfluidic screening systems enable high-throughput, labor-saving enzyme directed evolution by employing fluorescence, absorbance, and Raman-activated sorting strategies for library screening. Forster resonance energy transfer (FRET) - a nanoscale technique for monitoring intramolecular/intermolecular conformational changes - is yet to be integrated into this process. We upgraded a single-channel sorter to a dual-channel one without redesigning the microscopy setup, which can monitor FRET signals during enzyme reactions in droplets at kilohertz rates. We applied this upgraded sorter to improve the incorporation efficiency of KOD DNA polymerase towards reversible terminator deoxyribonucleotides, a property crucial for its application in next-generation sequencing (NGS). Our data show that a single-round sorting can achieve 30-fold enrichment of active variants. Five KOD variants enabling 100-cycle single-end runs of DNA sequencing were identified using a novel cyclic reversible termination (CRT) substrate featuring a terminator group {approx} 5-fold bulkier than the classical azidomethyl moiety. We also engineered a metagenome-derived novel polymerase, and a variant achieving 90% terminator incorporation efficiency within 2 minutes was identified after two rounds of enrichment. In sum, we provide a practical setup for dual-channel FRET-based droplet sorting, and demonstrate its ability in terminator polymerases engineering, thereby broaden the scope of microfluidic applications.

biochemistry↗

bioRxiv2024

Organ-specific Sympathetic Innervation Defines Visceral Functions

The autonomic nervous system orchestrates the brain and body functions through the sympathetic and parasympathetic pathways. However, our understanding of the autonomic system, especially the sympathetic system, at the cellular and molecular levels is severely limited. Here, we show unique topological representations of individual visceral organs in the major abdominal sympathetic ganglion complex. Using multi-modal transcriptomic analyses, we identified distinct sympathetic populations that are both molecularly and spatially separable in the celiac-superior mesenteric ganglia (CG-SMG). Notably, individual CG-SMG populations exhibit selective and mutually exclusive axonal projections to visceral organs, targeting either the gastrointestinal (GI) tract or secretory areas including the pancreas and bile tract. This combinatorial innervation pattern suggests functional segregation between different CG-SMG populations. Indeed, our neural perturbation experiments demonstrated that one class of neurons selectively regulates GI food transit. Another class of neurons controls digestion and glucagon secretion independent of gut motility. These results reveal the molecularly diverse sympathetic system and suggest modular regulations of visceral organ functions through distinct sympathetic populations.

neuroscience↗

bioRxiv2022

Zn2+ potentiation of OTOP proton channels identifies structural elements of the gating apparatus

Otopetrin proteins (OTOPs) form proton-selective ion channels that are expressed in diverse cell types where they may mediate detection of acids or regulation of pH. In vertebrates there are three family members: OTOP1 is required for formation of otoconia in the vesibular system and it forms the receptor for sour taste, while the functions of OTOP2, and OTOP3 are not yet known. Importantly, the gating mechanisms of any of the OTOP channels are not well-understood, and until recently, it was not even known if the channels were gated. Here we show that Zn2+, as well as other transition metals including copper (Cu2+), potently activate murine OTOP3. Zn2+ pre-exposure increases the magnitude of OTOP3 currents to a subsequent acid stimulus by as much as 10-fold. In contrast, OTOP2 currents are insensitive to potentiation by Zn2+. Swapping the extracellular tm 11-12 linker between OTOP3 and OTOP2 was sufficient to eliminate Zn2+ potentiation of OTOP3 and confer Zn2+ potentiation on OTOP2. We also show that H531 within the tm 11-12 linker is essential for potentiation of OTOP3 by Zn2+, likely by forming part of its binding site. Kinetic modeling of the data is consistent with Zn2+ stabilizing the open state of the channel, possibly competing with H+ for activation of the channels. These results establish the tm 11-12 linker as part of the gating apparatus of OTOP channels and a target for drug discovery. Zinc is an essential micronutrient and its regulation of OTOP channels will undoubtedly have important physiological sequelae. Significance StatementA family of proton-activated H+ ion channels was recently identified that includes the sour receptor OTOP1. Here we show that members of the OTOP channel family are differentially sensitive to Zn2+, which strongly activates OTOP3 but not OTOP2. By studying chimeric channels, we identify structural elements in the extracellular linker between transmembrane domains 11-12 that are necessary and sufficient for Zn2+ activation of OTOP channels. In addition to the taste system, OTOP channels play important roles in biomineralization in both vertebrate and invertebrates and are expressed in the digestive tract where their expression is a predictor of cancer prognosis. Our identification of the tm 11-12 linker as part of the gating apparatus makes it a promising target for pharmaceutical discovery.

biophysics↗

bioRxiv2022

Response to immune checkpoint blockade improved in pre-clinical model of breast cancer after bariatric surgery

Bariatric surgery is becoming more prevalent as a sustainable weight loss approach, with vertical sleeve gastrectomy (VSG) being the first line of surgical intervention. We and others have shown that obesity exacerbates tumor growth while diet-induced weight loss impairs obesity-driven progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters responses to therapy. Using a pre-clinical model of diet induced obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy was investigated. Weight loss by bariatric surgery or weight matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as dietary intervention in reducing tumor burden despite achieving a similar extent of weight and adiposity loss. Circulating leptin did not associate with changes in tumor burden. Uniquely, tumors in mice that received VSG displayed elevated inflammation and immune checkpoint ligand, PD-L1. Further, mice that received VSG had reduced tumor infiltrating T lymphocytes and cytolysis suggesting an ineffective anti-tumor microenvironment. VSG-associated elevation of PD-L1 prompted us to next investigate the efficacy of immune checkpoint blockade in lean, obese, and formerly obese mice that lost weight by VSG or weight matched controls. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden.

cancer biology↗

bioRxiv2022

Structural motifs for subtype-specific pH-sensitive gating of vertebrate otopetrin proton channels

Otopetrin (OTOP) channels are proton-selective ion channels conserved among vertebrates and invertebrates and with no structural similarity to other ion channels. There are three vertebrate OTOP channels (OTOP1, OTOP2, and OTOP3), of which one (OTOP1), functions as a sour taste receptor. Whether OTOP channels are gated by, as well as permeating, protons was not known. Here, by comparing functional properties of the three vertebrate proton channels with patch-clamp recording and cytosolic pH microfluorimetry, we provide evidence that each is gated by external protons. OTOP1 and OTOP3 are both activated by extracellular protons, with a sharp threshold of pHe <6.0 and 5.5 respectively, while OTOP2 is negatively gated by protons, and more conductive at alkaline extracellular pH (>pH 9.0). Strikingly, we found that we could change pH-sensitive gating of OTOP2 and OTOP3 channels by swapping extracellular linkers that connect transmembrane domains. Swaps of linkers within the N domain changed the relative conductance at alkaline pH, while swaps within the C domain tended to change the rates of OTOP3 current activation. We conclude that members of the OTOP channel family are proton-gated (acid-sensitive) proton channels and that the gating apparatus is distributed across multiple extracellular regions within both the N and C domains of the channels. In addition to the taste system, OTOP channels are found in the vestibular and digestive systems, where pH sensitivity may be tuned to specific functions.

biophysics↗

bioRxiv2021

Dynamic Detection and Reversal of Myocardial Ischemia Using an Artificially Intelligent Bioelectronic Medicine

SummaryMyocardial ischemia is spontaneous, usually asymptomatic, and contributes to fatal cardiovascular consequences. Importantly, biological neural networks cannot reliably detect and correct myocardial ischemia on their own. In this study, we demonstrate an artificially intelligent and responsive bioelectronic medicine, where an artificial neural network (ANN) supplements biological neural networks enabling reliable detection and correction of myocardial ischemia. ANNs were first trained to decode spontaneous cardiovascular stress and myocardial ischemia with an overall accuracy of [~]92%. ANN-controlled vagus nerve stimulation (VNS) reversed the major biomarkers of myocardial ischemia with no side effects. In contrast, open-loop VNS or ANN-controlled VNS following a caudal vagotomy essentially failed to reverse correlates of myocardial ischemia. Lastly, variants of ANNs were used to meet clinically relevant needs, including interpretable visualizations and unsupervised detection of emerging cardiovascular stress states. Overall, these results demonstrate that ANNs can supplement deficient biological neural networks via an artificially intelligent bioelectronic medicine system.

bioengineering↗

bioRxiv2019

Structures of the Otopetrin Proton Channels Otop1 and Otop3

Otopetrins (Otop1-Otop3) comprise one of only two known eukaryotic proton-selective channel families. Otop1 is required for formation of otoconia and is a candidate mammalian sour taste receptor. Here, we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture of Otopetrins with each subunit consisting of twelve transmembrane helices divided into structurally related N and C domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a cavernous central tunnel. Two hydrophilic vestibules, as well as the intrasubunit interface between N and C domains, form conduits for water entry into the membrane plane in molecular dynamics simulations, suggesting they each could provide pathways for proton conduction. We also demonstrate the functional relevance of a salt bridge in the C domain vestibule by mutagenesis. Our results provide a structural basis for understanding the function of the Otopetrin proton channel family.

biochemistry↗