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Biology subjects

Ejaz, A.

Publications and source records attributed to Ejaz, A..

9 recordsLinked to original sources

Rapid and reusable high-throughput microfluidics through modular assembly

High-throughput microfluidics has transformed biomedical research by enabling precise and parallel sample handling, but most devices are single-use due to channel occlusion and contamination from experiments. Alongside low fabrication yield and reduced experimental success associated with dense microfeatures, this creates a major bottleneck for scalable high-throughput applications. We present a rapid, reusable, and modular high-throughput microfluidic platform with integrated microvalves for automation. The platform employs a multilayer architecture consisting of a custom casing, PDMS layers with dense microfeatures for fluid handling and culture, and a glass substrate. Permanent bonding is applied only between control and fluid layers, while reversible bonding is used at all other interfaces, including the substrate. Because substrate is the primary cell-contact surface and can be readily detached, the remaining layers can be disassembled, thoroughly cleaned, and reused with minimal processing on a new substrate. This approach improves repeatability and experimental success while reducing preparation time from days to [~]2 hours. The disassemblable design also supports incorporation of application-specific layers between fluid layer and substrate, enhancing platform versatility for 3D culture. We validated performance through pressure/flow characterization and on-chip cell/organoid culture. Overall, our platform accelerates rapid high-throughput data generation across diverse biological applications.

bioengineering↗

A 3-genes interferon signature predicts sustained complete remission in pediatric AML patients

The immunological composition of the microenvironment has shown relevance for diagnosis, prognosis, and therapy in solid tumors but remains underexplored in acute leukemias. We investigated the significance of acute myeloid leukemia (AML) bone marrow microenvironment in predicting chemosensitivity and long-term remission in pediatric patients. We analyzed 32 non-promyelocytic pediatric AML patients at diagnosis using NanoString PanCancer IO 360 assay, RNA sequencing, and deep-phenotype flow cytometry analyses. The findings were validated using the pediatric TARGET AML dataset. A short signature of three interferon (IFN)-related genes (GBP1, PARP12, TRAT1) distinguished patients with chemosensitive disease and reduced minimal residual disease after induction chemotherapy. The signature stratified patients overall, and within the clinically defined "standard-risk" group, patients with high gene expression at diagnosis had significantly longer overall survival. The leukemia microenvironment associated with this signature showed enrichment of non-exhausted CD4+ and CD8+ T cytotoxic lymphocytes and expansion of CD8+ T effector memory cells re-expressing CD45RA (TEMRA) in patients with a favorable prognosis. Our results show the importance of the bone marrow microenvironment in pediatric AML and provide tools for a refined stratification of "standard-risk" patients, lacking adequate risk-oriented therapies. They also offer a promising guide for tackling immune pathways and exploiting immune-targeted therapies. Statement of significanceWe identified a novel three-gene IFN-related signature that distinguished pediatric AML patients by chemosensitivity and remission outcomes. It stratified patients across all risk groups, including the "standard-risk" group, with high expression linked to a T-cell-enriched microenvironment and longer survival. This signature may enhance risk stratification and guide targeted immunotherapy.

cancer biology↗

TWIST1 mediated transcriptional activation of SPON2 drives colorectal peritoneal metastasis through activation of cancer-associated fibroblast signaling network

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related mortality in the United States. Peritoneal metastasis (PM), a malignant dissemination within the peritoneal cavity, affects approximately 20% of CRC patients and accounts for 25-35% of stage IV cases. CRC PM is associated with dismal outcomes, with a median overall survival of only 16 months on systemic chemotherapy and an almost 0% five-year survival rate, largely due to frequent treatment resistance and limited therapeutic options. Despite advances in understanding CRC metastasis, the molecular mechanisms driving CRC PM remain poorly defined. CRC heterogeneity is classified into four Consensus Molecular Subtypes (CMS1-4), with CRC PM tumors predominantly exhibiting the CMS4 signature--characterized by stromal enrichment, high mesenchymal gene expression, and enhanced cellular plasticity--features linked to aggressive disease progression and resistance to standard chemotherapy. In this study, we identify TWIST1, a basic helix-loop-helix transcription factor, as significantly upregulated in CRC PM. We establish TWIST1-SPON2 as a novel transcriptional axis driving CRC PM tumorigenesis, mediating tumor-stroma interactions between tumor epithelium and cancer-associated fibroblasts (CAFs). Additionally, we identify SPP1, secreted by CAFs, as an upstream regulator of the TWIST1-SPON2 cascade via AKT activation in tumor cells. This newly defined SPP1-TWIST1-SPON2 signaling circuit plays a pivotal role in shaping the tumor microenvironment and promoting CRC PM progression. The findings establish the SPP1-TWIST1-SPON2 axis as a potential biomarker and a promising therapeutic target in CRC PM. Keyword: Colorectal cancer, peritoneal metastasis, epithelial-mesenchymal transition, cancer-associated fibroblast

cancer biology↗

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

Genomic Selection Pressure Discovery using Site-Frequency Spectrum & Reduced Local- Variability Statistics in Pakistani Dera-Din-Panah Goat

Population geneticists have long sought to comprehend various selection traces present in the goat genome due to natural or human-driven selection and breeding practices. As a step forward to pinpoint the selection signals in the Pakistani Dera-Din-Panah (DDP) goat breed, whole-genome pooled-sequencing (n=12) was performed and 618,236,192 clean paired-end reads were mapped against ARS1 reference goat assembly. Five different selection signal statistics were applied here using four Site-Frequency Spectrum (SFS) methods (Tajimas D (TD), Fay & Wus H (H), Zengs E (E), Pool - HMM) and one Reduced Local-Variability approach named pooled-heterozygosity (Hp). The under-selection regions were annotated with significant threshold values of -ZTD[≥]4.7, -ZH[≥]6, -ZE[≥]2.5, Pool-HMM[≥]12, and -ZHp[≥]5, which resulted in accumulative 364 candidate gene hits, while the highest signals were observed on Chr. 4, 6, 10, 12, 15, 16, 18, 20, 27 harbor ADAMTS6, CWC27 genes associated with body-height, RELN, MYCBP2, FGF14, STIM1, CFAP74, GNB1, CALML6, TMEM52, FAM149A, NADK, MMP23B, OPN3 with body-weight/meat production, FH, MFHAS1, KLKB1 with milk production, RRM1, KMO, SPEF2, F11 with fecundity rate/reproduction, ATP8B4 with immunity, KIT, KMO with pigmentation, ERI1 with olfaction and RHOG with wool production traits. Furthermore, we accentuate to highlight the putative windows that were captured commonly by any of the five statistical methods applied which harbor meat production, immunity and reproduction-associated genes validating the genotype-phenotype relationship of aforementioned traits private to this goat breed. Current insight into the genomic architecture of DDP goat provides a better understanding to improve its genetic potential and other vested traits of large body size and fiber production by updating the breeding strategies to boost the livestock-based agricultural economy of the country.

genetics↗

Differential Gene Expression Pipeline for Whole Transcriptome RNA-Seq Data using Personal Computer

Advances in the next generation sequencing (NGS) technologies, their cost effectiveness and well-developed pipelines using computational tools/softwares has allowed researchers to reveal ground-breaking discoveries in multi-omics data analysis. However, there is still uncertainty due to massive upsurge in parallel tools and difficulty in choosing best practiced pipeline for expression profiling of RNA sequenced (RNA-seq) data. Here, we detail the optimized pipeline that works at a fast pace with enhanced accuracy on personal computer rather than using cloud or high-performance computing clusters (HPC). The steps include quality check, base filtration, quasi-mapping, quantification of samples, estimation and counting of transcript/gene expression abundances, identification and clustering of differentially expressed features and visualization of the data. The tools FastQC, Trimmomatic, Salmon and some other scripts in Trinity toolkit were applied on two paired-end datasets. An extension of this pipeline may also be formulated in future for the gene ontology enrichment analysis and functional annotation of the differential expression matrix to make this data biologically more significant.

bioinformatics↗

Whole Genome Selective Sweeps Analysis in Pakistani Kamori Goat

Natural and artificial selection fix certain genomic regions of reduce heterozygosity which is an initial process in breed development. Primary goal of the current study is to identify these genomic selection signatures under positive selection and harbor genes in Pakistani Kamori goat breed. High throughput whole genome pooled-seq of Kamori (n = 12) and Bezoar (n = 8) was carried out. Raw fastq files were undergone quality checks, trimming and mapping process against ARS1 reference followed by calling variant allele frequencies. Selection sweeps were identified by applying pooled heterozygosity (Hp) and Tajimas D (TD) on Kamori while regions under divergent selection between Kamori & Bezoar were observed by Fixation Index (FST) analysis. Genome sequencing yielded 619,031,812 reads of which, 616,624,284 were successfully mapped. Total 98,574 autosomal selection signals were detected; 32,838 from Hp and 32,868 from each FST & TD statistics. Annotation of the regions with threshold (-ZHp [≥] 5, TD [≤] -2.72 & FST [≤] 0.09) detected 60 candidate genes. The top hits harbor Chr.1, 6, 8 & 21 having genes associated with body weight (GLIS3, ASTE1), coat color (DOCK8, MIPOL1) & body height (SLC25A21). Other significant windows harbor milk production, wool production, immunity, adaptation and reproduction trait related genes. Current finding highlighted the under-selection genomic regions of Kamori breed and likely to be associated with its vested traits and further useful in breed improvement, and may be also propagated to other undefined goat breeds by adopting targeted breeding policies to improve the genetic potential of this valued species.

genomics↗

Whole Genome Comparison of Pakistani Corona Virus with Chinese and US Strains along with its Predictive Severity of COVID-19

Recently submitted 784 SARS-nCoV2 whole genome sequences from NCBI Virus database were taken for constructing phylogenetic tree to look into their similarities. Pakistani strain MT240479 (Gilgit1-Pak) was found in close proximity to MT184913 (CruiseA-USA), while the second Pakistani strain MT262993 (Manga-Pak) was neighboring to MT039887 (WI-USA) strain in the constructed cladogram in this article. Afterward, four whole genome SARS-nCoV2 strain sequences were taken for variant calling analysis, those who appeared nearest relative in the earlier cladogram constructed a week time ago. Among those two Pakistani strains each of 29,836 bases were compared against MT263429 from (WI-USA) of 29,889 bases and MT259229 (Wuhan-China) of 29,864 bases. We identified 31 variants in both Pakistani strains, (Manga-Pak vs USA=2del+7SNPs, Manga-Pak vs Chinese=2del+2SNPs, Gilgit1-Pak vs USA=10SNPs, Gilgit1-Pak vs Chinese=8SNPs), which caused alteration in ORF1ab, ORF1a and N genes with having functions of viral replication and translation, host innate immunity and viral capsid formation respectively. These novel variants are assumed to be liable for low mortality rate in Pakistan with 385 as compared to USA with 63,871 and China with 4,633 deaths by May 01, 2020. However functional effects of these variants need further confirmatory studies. Moreover, mutated N & ORF1a proteins in Pakistani strains were also analyzed by 3D structure modelling, which give another dimension of comparing these alterations at amino acid level. In a nutshell, these novel variants are assumed to be linked with reduced mortality of COVID-19 in Pakistan along with other influencing factors, these novel variants would also be useful to understand the virulence of this virus and to develop indigenous vaccines and therapeutics.

genomics↗

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array \"waves\", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

genomics↗