Biology subjects

Shahid, S.

Publications and source records attributed to Shahid, S..

12 recordsLinked to original sources

bioRxiv2026

Mutational and bioinformatic analysis of the binding site for the ribonucleotide reductase-specific transcriptional repressor NrdR

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

molecular biology↗

bioRxiv2025

Glycoengineering of the hepatitis C virus E2 glycoprotein leads to improved biochemical properties and enhanced immunogenicity

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

immunology↗

bioRxiv2024

miRScore: a rapid and precise microRNA validation tool

MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression in many eukaryotes. Next-generation sequencing of small RNAs (small RNA-seq) has accelerated discovery and annotation of novel miRNAs. Newly discovered miRNAs are typically submitted to databases such as the miRBase microRNA registry following the publication of a peer-reviewed study. However, genome-wide scans using small RNA-seq data often yield high rates of false-positive miRNA annotations, highlighting the need for more robust validation methods. miRScore was developed as an independent and efficient tool for evaluating miRNA annotations using sRNA-seq data. miRScore combines structural and expression-based analyses to provide rapid and reliable validation of miRNA annotations. By providing users with detailed metrics and visualization, miRScore enhances the ability to assess confidence in novel and existing miRNA annotations. miRScore has the potential to advance the overall quality of miRNA annotations by improving accuracy of new submissions to miRNA databases and serving as a resource for re-evaluating existing annotations.

bioinformatics↗

bioRxiv2024

Experimental Warming Alters Nitrogen Cycle in a Humid Tropical Forest

O_LIHumid tropical forests typically contain high soil nitrogen (N), supporting rapid rates of primary productivity and recovery from disturbances. Nitrogen fixation is regulated by microbial communities, which can be free-living in the soil and leaf litter (asymbiotic) or in symbioses with plants. C_LIO_LITo investigate how warming affects asymbiotic and symbiotic components of the N cycle, we analyzed soil and leaf litter samples as well as annual seedling census data from an experimental warming field site in Puerto Rico. C_LIO_LI16S and nifH sequencing revealed that warming significantly altered bacterial composition. Asymbiotic N fixation rates were 55% greater in soil (0.004 nmol N2/g/hr) and 525% greater (0.217 nmol N2/g/hr) in leaf litter from warmed compared to ambient plots. This increase in fixation was associated with changes in the N-fixing bacterial community. Under warming, N fixers experienced a 4.4-fold increase in growth rate compared to non-fixers, yet competition with neighboring N fixers eventually reduced N fixer growth. Seedling growth, especially of N fixers, initially increased following hurricane disturbances before declining. C_LIO_LITogether, our findings suggest that warming increases the flux of N from the atmosphere into this tropical forest, driving changes in both microbial and tree dynamics. C_LI

ecology↗

bioRxiv2024

Bacterial transcriptional repressor NrdR - a flexiblemultifactorial nucleotide sensor

NrdR is a bacterial transcriptional repressor consisting of a Zn-ribbon domain followed by an ATP-cone domain. Understanding its mechanism of action could aid the design of novel antibacterials. NrdR binds specifically to two "NrdR boxes" upstream of ribonucleotide reductase operons, of which Escherichia coli has three: nrdHIEF, nrdDG and nrdAB, where we identified a new box. We show that E. coli NrdR (EcoNrdR) has similar binding strength to all three sites when loaded with ATP plus dATP or equivalent diphosphate combinations. No other combination of nucleotides promotes binding to DNA. We present crystal structures of EcoNrdR-ATP-dATP and EcoNrdR-ADP-dATP, which are the first high-resolution crystal structures of an NrdR. We have also determined cryo-EM structures of DNA-bound EcoNrdR-ATP-dATP and novel filaments of EcoNrdR-ATP. Tetrameric forms of EcoNrdR involve alternating interactions between pairs of Zn-ribbon domains and ATP-cones. The structures reveal considerable flexibility in relative orientation of ATP-cones vs Zn-ribbon domains. The structure of DNA-bound EcoNrdR-ATP-dATP shows that significant conformational rearrangements between ATP-cones and Zn-ribbons accompany DNA binding while the ATP-cones retain the same relative orientation. In contrast, ATP-loaded EcoNrdR filaments show rearrangements of the ATP-cone pairs and sequester the DNA-binding residues of NrdR such that they are unable to bind to DNA. Our results, in combination with a previous structural and biochemical study, point to highly flexible EcoNrdR structures that when loaded with the correct nucleotides adapt to an optimal promoter binding conformation.

biophysics↗

bioRxiv2024

Engineered NKG2C+ NK-like T cells exhibit superior antitumor efficacy while mitigating cytokine release syndrome

Engineered T and NK cell therapies have widely been used to treat hematologic malignancies and solid tumors, with promising clinical results. Current chimeric antigen receptor (CAR) T cell therapeutics have, however, been associated with treatment-related adverse events such as cytokine release syndrome (CRS) and are prone to immunologic exhaustion. CAR-NK therapeutics, while not associated with CRS, have limited in vivo persistence. We now demonstrate that an NK-like TCR{beta}+ CD8 T cell subset, identified and expanded ex vivo through its expression of the activating receptor NKG2C (NKG2C+ NK-like T cells), can be transduced to express a second-generation CD19 CAR (1928z), resulting in superior tumor clearance, longer persistence and decreased exhaustion compared to conventional 1928z CAR+ CD8 T cells and 1928z CAR+ NK cells. Moreover, CAR-modified NKG2C+ NK-like T cells resulted in significantly reduced CRS compared to conventional CAR+ CD8 T cells. Similarly, NKG2C+ NK-like T cells engineered with a TCR targeting the NY-ESO-1 antigen exhibit robust tumor control and minimal exhaustion compared to TCR-engineered conventional CD8 T cells. These data establish NKG2C+ NK-like T cells as a robust platform for cell engineering, and offer a safer, more durable alternative to conventional CAR-T and CAR-NK therapies.

immunology↗

bioRxiv2024

Strong conservation of spacer lengths in NrdR repressor DNA binding sites

The ribonucleotide reductase-specific repressor NrdR, from the human pathogens Listeria monocytogenes and Streptococcus pneumoniae, form tetramers that bind to DNA when loaded with dATP and ATP. If loaded with only ATP they form different oligomeric complexes that cannot bind to DNA. The DNA binding site in L. monocytogenes is a pair of NrdR boxes separated by 15-16 bp, whereas in Streptococcus pneumoniae the NrdR boxes are separated by 25-26 bp. However, Streptococcus pneumoniae NrdR binds stronger to the related Streptococcus thermophilus binding sites with NrdR boxes separated by 15-16 bp. This observation triggered a comprehensive binding study of four NrdRs from L. monocytogenes, Streptococcus pneumoniae, Escherichia coli and Streptomyces coelicolor to a series of synthetic dsDNA fragments where the NrdR boxes were separated by 12-27 bp. All four NrdRs bound well to NrdR boxes separated by 14-17 bp, and also to NrdR boxes separated by 24-27 bp. The worst binding occurred when NrdR boxes were separated by 20 bp. The in vitro results were confirmed in vivo in E. coli for spacer distances 12-27 bp. We conclude that NrdR repressors bind most efficiently when there is an integer number of DNA turns between the center of the two NrdR boxes.

biochemistry↗

bioRxiv2022

TCRi: Information theoretic metrics for single cell RNA and TCR sequencing in cancer

Single-cell T cell repertoire sequencing can pair both T cell receptor (TCR) and gene expression sequence data, providing an enriched view of T cell behavior. This powerful tool can identify and characterize specific clonotypes and phenotypes as well as track their changes in response to therapy, such as immune checkpoint blockade (ICB). We present a novel information theoretic framework called TCRi for characterizing single cell T cell repertoires by formalizing the relationship between clonotype and phenotype in a joint probability distribution. Our strategy allows for the identification of subpopulations of T cells and jointly quantifies their TCR and expression profiles in response to stimuli, in addition the framework tracks the phenotypic changes in individual T cell clones over time. We applied this framework to four datasets of T cells sequenced from cancer patients treated with anti-PD-(L)1 ICB immunotherapies and examined evolution of T cell responses pre- and post-treatment. Quantitative of phenotypic and clonotypic entropy analysis with TCRi demonstrated improvements in characterization of the transcriptional signature of clonotypes. Furthermore, TCRi highlighted the importance of phenotypic flux and specific T-cell phenotypes as determinants of therapeutic response.

bioinformatics↗

bioRxiv2020

SARS-CoV-2 genome analysis of strains in Pakistan reveals GH, S and L clade strains at the start of the pandemic

ObjectivesPakistan has a high infectious disease burden with about 265,000 reported cases of COVID-19. We investigated the genomic diversity of SARS-CoV-2 strains and present the first data on viruses circulating in the country. MethodsWe performed whole-genome sequencing and data analysis of SARS-CoV-2 eleven strains isolated in March and May. ResultsStrains from travelers clustered with those from China, Saudi Arabia, India, USA and Australia. Five of eight SARS-CoV-2 strains were GH clade with Spike glycoprotein D614G, Ns3 gene Q57H, and RNA dependent RNA polymerase (RdRp) P4715L mutations. Two were S (ORF8 L84S and N S202N) and three were L clade and one was an I clade strain. One GH and one L strain each displayed Orf1ab L3606F indicating further evolutionary transitions. ConclusionsThis data reveals SARS-CoV-2 strains of L, G, S and I have been circulating in Pakistan from March, at the start of the pandemic. It indicates viral diversity regarding infection in this populous region. Continuing molecular genomic surveillance of SARS-CoV-2 in the context of disease severity will be important to understand virus transmission patterns and host related determinants of COVID-19 in Pakistan.

genomics↗

bioRxiv2020

In vitro hydroxyapatite formation of a TTCP and DCPA based dentine desensitiser

The objective of this study was to characterise apatite formation ability of a tetra calcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) based desensitising product, Teethmate (TM). Methods: Cylindrical samples of TM were produced and immersed in TRIS buffer and artificial saliva (AS) for selected time periods. This was followed by characterisation of the phases formed using 31P Magic Angle - Nuclear Magnetic Resonance Spectroscopy, X-ray powder diffraction and Fourier-transform infrared spectroscopy. Dentine discs treated with the material were analysed by scanning electron microscopy (SEM) to evaluate crystal shape and tubules occlusion. Results: SEM analysis showed the formation of plate-like crystals occluding the dentinal tubules. Solid-state characterisation analyses showed that TM cement samples immersed in TRIS-buffer (pH: 7.3) formed hydroxyapatite phases within approximately six hours of immersion. HA formation was less rapid for the samples stored in AS (pH: 6.5). Conclusions: This study shows that a TTCP-DCPA based desensitising product (TM) converts to HA in vitro and provides an effective option for the treatment of dentine hypersensitivity.

biophysics↗

bioRxiv2020

In vitro hydroxyapatite formation of a TTCP and DCPA based dentine desensitiser

The objective of this study was to characterise apatite formation ability of a tetra calcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) based desensitising product, TeethmateTM (TM). Methods: Cylindrical samples of TM were produced and immersed in TRIS buffer and artificial saliva (AS) for selected time periods. This was followed by characterisation of the phases formed using 31P Magic Angle - Nuclear Magnetic Resonance Spectroscopy, X-ray powder diffraction and Fourier-transform infrared spectroscopy. Dentine discs treated with the material were analysed by scanning electron microscopy (SEM) to evaluate crystal shape and tubules occlusion. Results: SEM analysis showed the formation of plate-like crystals occluding the dentinal tubules. Solid-state characterisation analyses showed that TM cement samples immersed in TRIS-buffer (pH: 7.3) formed hydroxyapatite phases within approximately six hours of immersion. HA formation was less rapid for the samples stored in AS (pH: 6.5). Conclusions: This study shows that a TTCP-DCPA based desensitising product (TM) converts to HA in vitro and provides an effective option for the treatment of dentine hypersensitivity.

biophysics↗

bioRxiv2017

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs

First paragraphDodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite1, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production2. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation levels. Hosts with mutations in two of the targets supported significantly higher growth of C. campestris. Homologs of target mRNAs from diverse plants also have predicted target sites to induced C. campestris miRNAs, and the same miRNAs are expressed and active against host targets when C. campestris parasitizes a different host, Nicotiana benthamiana. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.

plant biology↗