Biology subjects

Shah, B.

Publications and source records attributed to Shah, B..

10 recordsLinked to original sources

bioRxiv2026

Discovery of novel antimicrobial resistance genes in food and fertiliser using a high-throughput gene capture and functional screening platform

Integrons are genetic elements that drive bacterial adaptation by capturing and expressing mobile gene cassettes. They play a key role in dissemination of antimicrobial resistance (AMR) genes, particularly in Gram-negative bacteria. In addition to known AMR determinants, integron gene cassettes carry a vast reservoir of novel genes whose functions are largely uncharacterised, making it diNicult to assess their full contribution to the resistome. Contributing to this are limitations in current sequence-based prediction methods which often lack the ability to identify unknown AMR or other adaptive genes with novel mechanisms. To address this, we developed an integron gene cassette capture system, a functional screening platform that captures environmental gene cassettes for direct phenotypic testing. Using this system, we recovered previously unknown AMR determinants while also providing insights into the prevalence of known clinical AMR genes in a range of environmental samples, including food items. Here we provide experimental data on multiple novel bleomycin resistance genes and a stress response gene conferring gentamicin and tobramycin resistance. Our sequence analysis of the captured library also highlighted the diversity of the environmental cassette pool, with 656 unique cassettes recovered, the majority of which encoded proteins with unknown functions. The cassette capture system is a powerful tool for accessing hidden elements of the resistome and discovering novel adaptive genes that may go undetected using current sequence-based approaches. Environmental implicationAntimicrobial resistance (AMR) genes are hazardous biological contaminants, yet the vast majority of environmental integron gene cassettes remain functionally uncharacterised. This study addresses this sequence-to-function gap by deploying a novel functional capture platform directly on realistic environmental matrices, including agricultural fertilisers, coastal seawater, and commercial food products. By characterising these cassettes, we uncovered hidden reservoirs of both novel and clinically established AMR genes circulating in critical exposure pathways. This work reveals the true hazardous potential of the mobile environmental resistome, validating a proactive One Health surveillance tool for monitoring emerging biological threats.

microbiology↗

bioRxiv2025

Effects of Cholesterol on Amyloid-induced Membrane Poration

The aggregation of amyloid peptides and their interactions with lipid membranes are central to the pathology of several neurodegenerative diseases. Using all-atom molecular dynamics simulations, we investigate how varying cholesterol concentrations (0%, 15%, and 30%) modulate amyloid-induced membrane poration. In cholesterol-free bilayers, pore formation was reproducibly observed in all simulations, whereas the presence of 15% cholesterol significantly reduced pore incidence, and 30% cholesterol entirely suppressed pore formation. Analysis revealed that cholesterol stiffens the bi-layer and strongly inhibits peptide-induced perturbations. Furthermore, cholesterol reduced the inter-leaflet mechanical coupling critical for transmembrane {beta}-sheet formation, a key step in pore initiation. Our findings suggest that increasing cholesterol content protects membranes against amyloid-induced poration, providing a potential molecular basis for the observed reduction in amyloid toxicity in cholesterol-enriched environments. These results offer new insights into the complex role of membrane com-position in amyloid-related diseases and highlight cholesterol as a potential modulator of amyloid cytotoxicity.

biochemistry↗

bioRxiv2025

Small-molecule allosteric activator of ubiquitin-specific protease 7 (USP7)

Ubiquitin-specific protease 7 (USP7) is a deubiquitylase essential for cell homeostasis, DNA repair, and regulation of both tumor suppressors and oncogenes. Inactivating USP7 mutations have been associated with Hao-Fountain Syndrome (HAFOUS), a rare neurodevelopmental disorder. Although a range of USP7 inhibitors have been developed over the last decade, in the context of HAFOUS as well as oncogene regulation, USP7 activators may represent a more relevant approach. To address this challenge, we report the discovery and characterization of a small-molecule activator of USP7 called MS-8. We showed that MS-8 activates USP7 by engaging the allosteric C-terminal binding pocket of USP7, thus mimicking the allosteric autoactivation by the USP7 C-terminal tail. We observed that MS-8 engages and activates mutant USP7 in a cellular context, impacting downstream proteins. Taken together, our study provides validation of the USP7 activator that paves the way towards novel activation-driven USP7 pharmacology.

biochemistry↗

bioRxiv2024

Structure-guided engineering of α-ketoisocaproate dioxygenase increases isobutene production in Synechocystis sp. PCC 6803

Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the -ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with {rho}-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with -ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC and elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis. HighlightsO_LIPromiscuous -ketoisocaproate dioxygenase (KICD) from Rattus norvegicus was overproduced in Synechocystis sp. PCC 6803. C_LIO_LIProtein engineering was used to improve substrate selectivity. C_LIO_LIThe generated F336V variant led to a 4-fold increase in isobutene production in vivo. C_LI

bioengineering↗

bioRxiv2024

Ternary complex formation of VCP, VCPIP1 and p47 facilitates Golgi reassembly

VCP/p97 regulates a wide range of cellular processes, including post-mitotic Golgi reassembly. In this context, VCP is assisted by p47, an adapter protein, and VCPIP1, a deubiquitinase (DUB). However, how they organize into a functionally competent complex and promote reassembly remains unknown. Here, we use cryo-EM to characterize VCP-VCPIP1 and VCP-VCPIP1-p47 complexes. We show that VCPIP1 engages VCP through two interfaces: one involving the N-domain of VCP and the UBX domain of VCPIP1, and the other involving the VCP D2 domains and a region of VCPIP1 we refer to as VCPID. The p47 UBX domain competitively binds to the VCP N-domain, while not affecting VCPID binding. We show that VCPID is critical for VCP-mediated enhancement of DUB activity and Golgi reassembly.

biochemistry↗

bioRxiv2024

Reading Outside the Lines: A Systematic Approach for Detecting Bias in Scientific Communication

Consciousness of the social impact of science and the potential biases of its authors is critical to understanding, interpreting, and using scientific findings responsibly. This is especially true for sciences concerned with human health and behavior, where societal and unconscious biases may reinforce existing inequities and discriminatory practices. Considering backgrounds and biases, we may notice bias influencing scientists methodological choices and conclusions, even when a work is otherwise scientifically sound. To this end, we created the pedagogical tool Finding inEquity in LIterature and eXperimentation (FELIX). FELIX is a tool that systematizes the detection of bias and subjectivity in scientific communications by using a three-phase progression of (i) Annotation, (ii) Analysis and (iii) Synthesis, where students form a unified argument about the text with a focus on its relationship to social or ethical context. Results from a mixed methods approach indicated the efficacy of our approach in supporting student learning related to reading comprehension, critical thinking skills, and in understanding the social and ethical implications of the research they were reading. We put forward FELIX as a universal method for training students in the reading of scientific communications and as a tool for addressing systemic inequities in science and science education.

scientific communication and education↗

bioRxiv2023

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.

synthetic biology↗

bioRxiv2023

AedG, a new weapon in Acinetobacters fight against antimicrobials

A novel multidrug efflux pump, AadT from the Drug:H+ antiporter 2 family, was discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential and examined the distribution of this gene. Putative homologs of this efflux pump were encoded in many Acinetobacter species and other Gram-negative species, and were genetically associated with novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump conferred decreased susceptibility to at least eight diverse antimicrobials, including antibiotics erythromycin, tetracycline; biocides chlorhexidine; and dyes ethidium bromide and DAPI. These results show that AadT is a new determinant in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).

microbiology↗

bioRxiv2021

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical -amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.{Delta}A.opt and C321.{Delta}A.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural -amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

synthetic biology↗

bioRxiv2018

The structural basis for release factor activation during translation termination revealed by time-resolved cryogenic electron microscopy

When the mRNA translating ribosome encounters a stop codon in its aminoacyl site (A site), it recruits a class-1 release factor (RF) to induce hydrolysis of the ester bond between peptide chain and peptidyl-site (P-site) tRNA. This process, called termination of translation, is under strong selection pressure for high speed and accuracy. Class-1 RFs (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have structural motifs that recognize stop codons in the decoding center (DC) and a universal GGQ motif for induction of ester bond hydrolysis in the peptidyl transfer center (PTC) 70 [A] away from the DC. The finding that free RF2 is compact with only 20 [A] between its codon reading and GGQ motifs came therefore as a surprise1. Cryo-electron microscopy (cryo-EM) then showed that ribosome-bound RF1 and RF2 have extended structures2,3, suggesting that bacterial RFs are compact when entering the ribosome and switch to the extended form in a stop signal-dependent manner3. FRET4, cryo-EM5,6 and X-ray crystallography7, along with a rapid kinetics study suggesting a pre-termination conformational change on the millisecond time-scale of ribosome-bound RF1 and RF28, have lent indirect support to this proposal. However, direct experimental evidence for such a short-lived compact conformation on the native pathway to RF-dependent termination is missing due to its transient nature. Here we use time-resolved cryo-EM9,10,11,12,13 to visualize compact and extended forms of RF1 and RF2 at 3.5 and 4 [A] resolution, respectively, in the codon-recognizing complex on the pathway to termination. About 25% of ribosomal complexes have RFs in the compact state at 24 ms reaction time after mixing RF and ribosomes, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

biophysics↗