Biology subjects

Siddique, A.

Publications and source records attributed to Siddique, A..

9 recordsLinked to original sources

bioRxiv2026

3D-Printed Scaffolds for Local Chemotherapeutic Delivery in Resected Spine Metastases

Spinal metastases often occur secondary to breast, lung and prostate cancer and lead to instability, pain and poor quality of life. Standard care for spine metastases includes a multidisciplinary approach with surgery playing a major role in tumor resection, stabilization and decompression. Surgical resection with adjuvant is an effective treatment, yet it is often accompanied by tumor recurrence from residual disease. Furthermore, acrylic cements applied to defect sites provide stability, but they do not promote bone repair and can become destabilized during recurrence events. Developing new tools to stabilize defect sites, promote bone repair and locally deliver therapeutics may circumvent these limitations. We have previously developed mechanically competent 3D printed lactide/mineral scaffolds conducive to bone repair in vivo. We have also developed 3D printed nanoporous scaffolds conducive to both bone repair and chemotherapeutic delivery. Here, we set out to assess doxorubicin and cisplatin uptake and release rates and efficacy of drug delivery in 2D and custom physiological 3D cultures of two human cancer cell lines associated with metastases, MDA-MB-231 (human breast) and C42B (human prostate). Composite scaffolds had a compressive modulus close to trabecular bone, and could sustainably and effectively release doxorubicin and cisplatin as measured against both breast and prostate cell lines in 2D and 3D custom physiological metastases models. As a proof-of-concept, doxorubicin loaded composite scaffolds were implanted into rat caudal vertebrae following MDA-MB-231 xenograft resection. Following 6 weeks of implantation, no adverse events were noted and microCT analysis revealed boney integration of the construct. Taken together, these data indicate that our composite scaffolds may be an appropriate alternate therapy to stabilize bone defects, promote bone repair and effectively inhibit cancer recurrence post-tumor resection. Future work will test composite scaffolds using in vivo bone metastases models.

bioengineering↗

bioRxiv2024

deepBlastoid: A Deep Learning-Based High-Throughput Classifier for Human Blastoids Using Brightfield Images with Confidence Assessment

Recent advances in human blastoids have opened new avenues for modeling early human development and implantation. Human blastoids can be generated in large numbers, making them suitable for high-throughput screening, which often involves analyzing vast numbers of images. However, automated methods for evaluating and characterizing blastoid morphology are still underdeveloped. We developed a deep-learning model capable of recognizing and classifying blastoid brightfield images into five distinct quality categories. The model processes 53.2 images per second with an average accuracy of 87%, without signs of overfitting or batch eHects. By integrating a Confidence Rate (CR) metric, the accuracy was further improved to 97%, with low-CR images flagged for human review. In a comparison with human experts, the model matched their accuracy while significantly outperforming them in throughput. We demonstrate the value of the model in two real-world applications: (1) systematic assessment of the eHect of lysophosphatidic acid (LPA) concentration on blastoid formation, and (2) evaluating the impact of dimethyl sulfoxide (DMSO) on blastoids for drug screening. In the applications involving over 10,000 images, the model identified significant eHects of LPA and DMSO, which may have been overlooked in manual assessments. The deepBlastoid model is publicly available and researchers can train their own model according to their imaging conditions and blastoid culture protocol. deepBlastoid thus oHers a precise, automated approach for blastoid classification, with significant potential for advancing mechanism research, drug screening, and clinical in vitro fertilization (IVF) applications.

developmental biology↗

bioRxiv2024

Aberrant expression of collagen type X in solid tumor stroma is associated with EMT, immunosuppressive and pro-metastatic pathways, bone marrow stromal cell signatures, and poor survival prognosis

BackgroundCollagen type X (ColX1, encoded by COL10A1) is expressed specifically in the cartilage-to-bone transition, in bone marrow cells, and in osteoarthritic (OA) cartilage. We have previously shown that ColX1 is expressed in breast tumor stroma, correlates with tumor-infiltrating lymphocytes, and predicts poor adjuvant therapy outcomes in ER+/HER2+ breast cancer. However, the underlying molecular mechanisms for these effects are unknown. In this study, we performed bioinformatic analysis of COL10A1-associated gene modules in breast and pancreatic cancer as well as in cells from bone marrow and OA cartilage. These findings provide important insights into the mechanisms of transcriptional and extracellular matrix changes which impact the local stromal microenvironment and tumor progression. MethodsImmunohistochemistry was performed to examine collagen type X expression in solid tumors. WGCNA was used to generate COL10A1-associated gene networks in breast and pancreatic tumor cohorts using RNA-Seq data from The Cancer Genome Atlas. Computational analysis was employed to assess the impact of these gene networks on development and progression of cancer and OA. Data processing and statistical analysis was performed using R and various publicly-available computational tools. ResultsExpression of COL10A1 and its associated gene networks highlights inflammatory and immunosuppressive microenvironments, which identify aggressive breast and pancreatic tumors and contribute to metastatic potential in a sex-dependent manner. Both cancer types are enriched in stroma, and COL10A1 implicates bone marrow-derived fibroblasts as drivers of the epithelial-to-mesenchymal transition (EMT) in these tumors. Heightened expression of COL10A1 and its associated gene networks is correlated with poorer patient outcomes in both breast and pancreatic cancer. Common transcriptional changes and chondrogenic activity are shared between cancer and OA cartilage, suggesting that similar microenvironmental alterations may underlie both diseases. ConclusionsCOL10A1-associated gene networks may hold substantial value as regulators and biomarkers of aggressive tumor phenotypes with implications for therapy development and clinical outcomes. Identification of tumors which exhibit high expression of COL10A1 and its associated genes may reveal the presence of bone marrow-derived stromal microenvironments with heightened EMT capacity and metastatic potential. Our analysis may enable more effective risk assessment and more precise treatment of patients with breast and pancreatic cancer. Research HighlightsO_LIColX highlights features of EMT in breast and pancreatic cancer C_LIO_LIColX gene modules are immunosuppressive and pro-metastatic C_LIO_LIColX-associated gene networks contribute to sex differences in pancreatic cancer C_LIO_LIColX-positive fibroblasts define more aggressive tumors with poorer survival C_LIO_LIColX is emerging as a biomarker for bone marrow-derived cells in cancer C_LI

cancer biology↗

bioRxiv2024

Avian-restrict Salmonella transition to endemicity is accompanied by localized resistome adaptation

Bacterial regional demonstration after global dissemination is an essential pathway for selecting distinct finesses. However, the evolution of the resistome during the transition to endemicity remains unaddressed. Using the most comprehensive whole-genome sequencing dataset of Salmonella enterica serovar Gallinarum (S. Gallinarum) collected from 15 countries, including 45 newly recovered samples from two related local regions, we established the relationship among avian-specific pathogen genetic profiles and localization patterns. Initially, we revealed the international transmission and evolutionary history of S. Gallinarum to recent endemicity through phylogenetic analysis conducted using a spatiotemporal Bayesian framework. Our findings indicate that the independent acquisition of the resistome via the mobilome, primarily through plasmids and transposons shapes a unique antimicrobial resistance profile among different lineages. Notably, the mobilome-resistome combination among distinct lineages exhibits a geographical-specific manner, further supporting a localized endemic mobilome-driven process. Collectively, this study elucidates resistome adaptation in the endemic transition of an avian-specific pathogen, likely driven by the localized farming style, and provides valuable insights for targeted interventions.

microbiology↗

bioRxiv2024

Lacticaseibacillus rhamnosus P118 enhances host tolerance to Salmonella infection by promoting microbe-derived indole metabolites

Salmonella is one of the most common foodborne pathogens, resulting in inflammatory gastroenteritis and frequently accompanied by dysbiosis. Gut commensals, such as Lactobacillus species, have been proven to exhibit broad anti-bacterial activities and protect hosts against pathogenic infections. Here, Lacticaseibacillus rhamnosus strain P118, screened from 290 isolates recovered from fermented yoghurts and healthy piglet intestines using traditional and Caenorhabditis elegans-infection screening strategies, exerts great probiotic properties. Notably, P118 and its supernatant exhibited great antibacterial activities and attenuated C. elegans susceptibility to Salmonella infection. We found that P118 protected mice against Salmonella lethal infections by enhancing colonization resistance, reducing pathogen invasion, alleviating intestinal pro-inflammatory response, and improving microbial dysbiosis and fecal metabolite changes. Microbiota and fecal metabolome analyses suggested P118 administration significantly decreased the relative abundances of potentially harmful microbes (e.g., Salmonella, Anaeroplasma, Klebsiella) and increased the fecal levels of tryptophan and its derivatives (indole, indole-3-acrylic acid, 5-hydroxytryptophan, 5-methoxyindoleacetate). Deterministic processes determined the gut microbial community assembly of P118-pretreated mice. Integrated omics further demonstrated that P118 probiotic activities in enhancing host tolerance to Salmonella infection were mediated by microbe-derived tryptophan/indole metabolites (e.g., indole-3-acrylic acid, indole, tryptophan, 5-methoxyindoleacetic acid, and 5-hydroxytryptophan). Collective results demonstrate that L. rhamnosus P118 could enhance host tolerance to Salmonella infections via various pathways, including direct antibacterial actions, inhibiting Salmonella colonization and invasion, attenuating pro-inflammatory responses of intestinal macrophages, and modulating gut microbiota mediated by microbe-derived indole metabolites.

microbiology↗

bioRxiv2023

Indigenous probiotics Lactobacillus reuteri and Enterococcus faecium exhibit positive growth performance and disease prevention against extended-spectrum cephalosporin and fluoroquinolones resistant Salmonella enterica in broiler chicks.

ABSTRACTThe rapid increase in antibiotic resistance poses a global threat to public health, necessitating the development of effective antimicrobial alternatives. This study compared an indigenous probiotic mix containing Lactobacillus reuteri and two strains of Enterococcus faecium to a commercial probiotic blend ProtexinR on the growth performance, mortality rate, histomorphology, serum immunoglobulins, and intestinal microflora of broiler chickens challenged with two multi drug resistant Salmonella serovars, Typhimurium and Enteritidis. Two hundred and forty day-old broiler chicks were randomly assigned to six treatment groups for 4 weeks: the treatment groups were; birds continuously supplemented with only indigenous probiotic strains (108 CFU/mL) (IPRO-); birds challenged with Salmonella serovars 106 (CFU/mL) (PC+); birds continuously supplemented with indigenous probiotic strains and challenged with Salmonella serovars (IPRO+); birds supplemented with ProtexinR and challenged with Salmonella serovars (CM+); birds supplemented with only ProtexinR (CM-); and birds with no Salmonella challenge or probiotics (negative control; PC-). The results revealed that IPRO- diets significantly improved feed conversion ratio (FCR) and increased body weight (BW) (P [≤] 0.05). No effect of probiotic treatments was observed on IPRO- and CM- on relative organ weights as compared to the negative control (PC-). The Salmonella- challenged group PC+ had the highest (20%) mortality rate and lowest BW. The IPRO- had significantly lower FCR (1.55) compared to PC- (1.86) and PC+ (1.95). The broilers in the IPRO- group showed significantly increased serum concentrations of IgA and IgG relative to both control groups (P [≤] 0.05). Morphological analysis of the ileum revealed significant increases (P [≤] 0.05) in the villus height and villus height/crypt depth in birds fed IPRO- compared with the PC+. Cecal Lactobacillus and Enterococcus counts were the highest (P [≤] 0.05) and Salmonella counts were the lowest (P [≤] 0.05) in the IPRO- group compared to the Salmonella infected group PC+. These results indicated that indigenous probiotic strains Lactobacillus reuteri and Enterococcus faecium can be an effective and low-cost alternative compared to commercial probiotics in the Pakistan poultry industry.

microbiology↗

bioRxiv2023

Nanoparticle-Functionalized Acrylic Bone Cement for Local Therapeutic Delivery to Spinal Metastases

Polymethylmethacrylate bone cement is often used to reconstruct critical-sized defects generated by surgical resection of spinal metastases. Residual tumor cells after a resection can drive recurrence and destabilization. Doxorubicin (DOX) is a common chemotherapeutic drug with unwanted side-effects when administered systemically. Mesoporous silica nanoparticles are gaining attention for targeted drug delivery to bypass the negative side effects associated with systemic drug administration. We developed a nanoparticle-functionalized cement for the local release of DOX and tested its ability to suppress cancer cells. DOX was loaded onto nanoparticles which were then mixed into the cement. Drug release profiles were obtained over a period of 4 weeks. Cement constructs were incubated with 2D and 3D cultures of breast and prostate cancer cell lines, and cell metabolic activity and viability were evaluated. Cell migration and spheroid growth were assessed in collagen-coated spheroid cultures. Nanoparticles were homogenously dispersed and did not alter cement mechanical strength. A sustained DOX release profile was achieved with the addition of nanoparticles to the bone cement. The release profile of DOX from nanoparticle cement may be modified by varying the amount of the drug loaded onto the nanoparticles and the proportion of nanoparticles in the cement. Cells treated with the cement constructs showed a dose- and time-dependent inhibition. Cell migration and spheroid growth were impaired in 3D culture. We show that nanoparticles are essential for sustained DOX release from bone cement. DOX-loaded nanoparticle cement can inhibit cancer cells and impair their migration, with strong potential for in vivo translation studies.

bioengineering↗

bioRxiv2022

A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics

Single-cell transcriptomics suffers from lapses in coverage of the full transcriptome, providing an incomplete gene expression profile of the cell. Here, we introduce single-cell CRISPRclean (scCLEAN), an in vitro molecular method that can be universally inserted into any single-cell RNA-seq workflow to improve the sensitivity of the assay. Utilizing CRISPR/Cas9, scCLEAN works to selectively remove highly abundant uninformative molecules, redistributing ~50% of reads to enrich for lowly expressed transcripts. Utilizing immune cells, we describe a validation of scCLEAN showing a 2.1-fold enrichment in library complexity with negligible off-target effects. Subsequently, applying scCLEAN to single-cell iso-seq samples results in a 4.6-fold improvement in unique isoform detection. Thus, demonstrating a benefit in short and long read sequencing applications. Finally, we illustrate the ability of scCLEAN to elucidate biological insights by applying it to two participant cohorts of cardiovascular samples, bringing to light novel molecular characteristics including inflammatory signatures.

genetics↗

bioRxiv2017

A Genome-wide Survey of Mutations in the Jurkat Cell Line

The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some irregularities were observed in Jurkats expression of central regulators of T cell receptor signaling, which raised doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm the majority of documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.

genomics↗