Biology subjects

Zafar, F.

Publications and source records attributed to Zafar, F..

13 recordsLinked to original sources

bioRxiv2026

APOE4 Drives Uniquely Dysfunctional Human Microglial States in Alzheimer's Disease

Variation in APOE, notably the {varepsilon}4 allele, profoundly shapes risk and severity of late-onset Alzheimers disease (AD), yet how it remodels human microglial states remains unresolved. We combine spatially resolved proteomic profiling with single-nuclear multiomic analyses to define microglial organization across APOE3/3 and APOE4/4 genotypes in AD. Quantifying condition-associated variation across the cellular manifold reveals a continuous landscape of microglial states. APOE4/4 shifts cells toward terminal states marked by loss of homeostatic identity, metabolic disruption, and incomplete acquisition of disease-associated programs. We identify an APOE4/4-enriched population in AD that exhibits inflammatory signaling without effective metabolic or phagocytic engagement, localizing to niches of gliosis and senescence, and coupled to chronic stress adaptation programs. Together with evidence that APOE4/4 potentiates the activation threshold of nascent microglia, these findings establish a unified framework for human microglial state change, linking genetic risk to spatial and molecular organization of immune responses in the AD brain. Graphical Abstract.APOE4/4 in Alzheimers disease reshapes microglial fate along continuous trajectories characterized by proteomic, transcriptional, and epigenetic programs consistent with chronic stress adaptation, alongside distinct composite spatial niches comprised of astrocytic gliosis and cellular senescence. O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/733295v1_ufig1.gif" ALT="Figure 1"> View larger version (75K): org.highwire.dtl.DTLVardef@b2afa4org.highwire.dtl.DTLVardef@12978f8org.highwire.dtl.DTLVardef@1c4e46dorg.highwire.dtl.DTLVardef@170e44d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Optical genome mapping identifies source-associated structural variant differences across early-passage human iPSCs

BackgroundInduced pluripotent stem cells (iPSCs) are an important model for studying human diseases in vitro. However, previous studies have shown that iPSC reprogramming and extended cell culture can introduce genomic structural variants (SVs). Technologies like karyotyping, CNV microarrays, and whole-genome sequencing have limitations in resolution, sensitivity, or the ability to detect large and complex structural variants compared to optical genome mapping (OGM). OGM is a genome-wide structural variant detection method that analyzes fluorescently labeled ultra-high-molecular-weight DNA molecules to identify copy-number and balanced rearrangements. At sufficient coverage, OGM can detect SVs at approximately [≥]2 kbp and identify mosaic events supported by molecule-level evidence, offering higher resolution than conventional karyotyping or SNP-array-based QC. Here, we compared iPSC clones derived from peripheral blood mononuclear cells (PBMCs) and fibroblasts (FBCs) to determine whether starting somatic cell source is associated with differences in structural variant burden and SV-type profiles after nuclear reprogramming into iPSCs. ResultsWe analyzed 73 low-passage iPSC clones generated from 25 parental lines using OGM. Compared with PBMC-iPSCs, FBC-iPSCs showed higher SV burden with the enrichment of duplications [≥]100 kbp, more frequent overlap with protein-coding genes, fragile sites, and recurrent chromosomal hotspot regions. In contrast, PBMC-iPSCs showed fewer SVs overall, and a higher proportion of clones without detectable clone-specific SVs. ConclusionsOGM provides a high-resolution approach for post-reprogramming genomic quality control by detecting clone-specific structural variants at approximately [≥]2 kbp, including events below the resolution of conventional cytogenetic and SNP-array-based assays. In these early passage iPSCs, SVs overlapped protein-coding genes, fragile sites, and recurrent culture-associated chromosomal regions, underscoring the need for clone-level genomic assessment before downstream applications. FBC-derived iPSCs showed a higher SV burden, including more frequent and larger duplications, whereas PBMC-derived iPSCs more often lacked detectable clone-specific SVs. These findings suggest that PBMC-iPSCs and FBC-iPSCs can differ in post-reprogramming SV profiles and support the use of OGM as a QC strategy during iPSC generation and selection.

genomics↗

bioRxiv2025

Utilizing Genomics to Identify Novel Immunotherapeutic Targets in Multiple Myeloma High-Risk Subgroups

Immunotherapy is now standard of care for multiple myeloma, where the most common targets are B cell maturation antigen, CD38, and G protein-coupled receptor class C group 5 member D but strategies to identify additional targets are needed. We have utilized two large datasets of genomic data and integrated them with existing databases to identify expressed cell surface targets in myeloma patients. Importantly, we also identify targets specific to genomic defined subgroups of patients including primary translocations and high-risk subgroups. Examples of subgroup targets include ROBO3 in t(4;14), CD109 in t(14;16), CD20 in t(11;14), GPRC5D in 1q+, and ADAM28 in biallelic TP53 samples. Expression was validated by flow cytometry and CRISPR-Cas9 knock out models. Sub-clonal differences in expression were noted, as was alternative splicing of existing immunotherapy targets such as FCRL5. These results highlight the use of genomic stratification to identify novel therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=157 HEIGHT=200 SRC="FIGDIR/small/635544v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@6965e6org.highwire.dtl.DTLVardef@1a0e567org.highwire.dtl.DTLVardef@34fc30org.highwire.dtl.DTLVardef@98c3d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Optical Genome Mapping of the human reference iPSC line KOLF2.1J reveals new smaller structural variants in neurodevelopmental genes

The INDI consortium curated the KOLF2.1J human iPSC line to create a reference cell line for neurological disease modeling. However, despite careful assessments, two separate studies found using SNP arrays identified five structural variants (SVs) with sizes >100kbp. Two heterozygous SVs overlap the genes JARID2, DTNBP1, and ASTN2, raising concerns about KOLF2.1Js suitability as a reference line. To investigate further, we screened KOLF2.1J for SVs smaller than 100kbp using optical genome mapping (OGM) to produce a high-resolution karyotype. OGM, validated by qPCR, indicated that one of the five known SVs contained a previously undetected overlap with RYBP. RYBP plays regulatory roles in neuronal differentiation, PAX6 expression and Notch signaling. Furthermore, OGM identified 11 SVs smaller than 100kbp, whose overlaps include the genes PER2, CSMD1, and PALS1. In summary, these mutations should be considered by researchers when using KOLF2.1J as a reference iPSC line for designing studies and experiments.

cell biology↗

bioRxiv2023

Robust nuclease-dead S. aureus dCas9-mediated alpha-synuclein knockdown in substantia nigra in a humanized mouse model of Parkinson's disease

Parkinsons disease (PD) is becoming increasingly prevalent due to an aging society, which places a substantial disease burden on patients and their families and an annual cost estimated at 52 billion dollars. However, no approved disease modulatory therapies that halt disease progression are available. Alpha-synuclein is a critical therapeutic target found in aggregated form in Lewy bodies which is the diagnostic hallmark of PD. Familial autosomal dominant forms of PD can present with causative exonic point mutations, copy number multiplications, and non-coding risk variants in the alpha-synuclein gene. The disease onset, severity, and progression depend on the gene expression levels of the alpha-synuclein. Here, we demonstrate that Streptococcus aureus dCas9 (sadCas9)-mediated CRISPR interference (CRISPRi) reduces alpha-synuclein mRNA and protein levels in a humanized mouse model. The mechanism of action is based on the principle that a complementary single guide RNA (sgRNA) recruits the sadCas9 protein to the promoter region of alpha-synuclein and modulates target gene transcription, leading to reduced gene expression. We show robust downregulation of alpha-synuclein in neurons after unilateral stereotactic injection into the substantia nigra of adult mice 1 and 6 months after surgery. This work shows proof of concept that viral-mediated sadCas9 CRISPR interference can be a promising therapeutic strategy to reduce alpha-synuclein in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556425v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9247org.highwire.dtl.DTLVardef@beb419org.highwire.dtl.DTLVardef@21e0f9org.highwire.dtl.DTLVardef@391d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisThis study focuses on the development of an AAV9 nuclease-dead S. aureus CRISPR/Cas9 expression system designed to target the human SNCA promoter. The objective is to achieve downregulation of -synuclein (a-syn) expression. After performing surgery and introducing the expression system, the levels of a-syn were measured at 1 month and 6 months post-surgery. The results indicate a significant downregulation of a-syn at both time points. Furthermore, it was observed that the initial immune response to the system attenuated over time, reaching control levels at the 6-month mark. These findings suggest the potential of this expression system for long-term downregulation of a-syn and provide insights into the immune response dynamics associated with its use.

neuroscience↗

bioRxiv2023

Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

neuroscience↗

bioRxiv2022

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice

We report on biallelic homozygous and monoallelic de-novo variants in SLITRK3 in 3 unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, we bring additional evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG, as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, our results exhibit impaired development of peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. Our study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

genetics↗

bioRxiv2022

Design And Optimization Studies Of Tablet In Tablet Formulation Of Diclofenac And Misoprostol: Application Of Response Surface Methodolgy And Compressional Behaviour Strategy

The present study was aimed to develop compression coated tablets of diclofenac sodium (75mg) in the inner core and misoprostol (200 mg) as the outer shell for the effective treatment of rheumatoid arthritis as this dosing frequency is not available in the market yet. Diclofenac sodium inner tablet was manufactured by wet granulation and its enteric coating was applied by Eudragit L 100-55, Isopropyl Alcohol and Opadry II Blue 85 F205034. While immediate-release misoprostol outer shell was also manufactured by wet granulation and coated by Opadry white and Polyethylene glycol 6000. Design of experiment(R) software was used for formulation design and optimization. Quality attributes such as tablet weight, hardness, disintegration time, percent drug dissolution and assay were performed as per official methods and satisfactory results were reported. Physical and chemical stability of selected formulations was evaluated following the ICH guidelines for accelerated stability testing. The compressional analysis of optimized formulation was performed to check the optimum compression pressure to obtain a stable formulation. Based on satisfactory quality attributes; formulation DF9MF7 was successfully developed as compression coated tablet with calculated shelf life of 4.8years. Compression coated tablets comprising of enteric coated diclofenac sodium as inner core and misoprostol as outer shell were successfully developed by wet granulation.

pharmacology and toxicology↗

bioRxiv2022

Multiplex imaging of human induced pluripotent stem cell-derived neurons with CO-Detection by indEXing (CODEX) technology

BackgroundHuman induced pluripotent stem cell (iPSC) models have been hailed as a breakthrough for understanding disease and developing new therapeutics. The major advantage of iPSC-derived neurons is that they carry the genetic background of the donor, and as such could be more predictive for clinical translation. However, the development of these cell models is time-consuming and expensive and it is thus critical to maximize biomarker readout from every model that is developed. One option is to use a highly multiplexed biomarker imaging assay, like CO-Detection by indEXing (CODEX), which allows detection of 50+ targets in situ at single-cell resolution. New MethodThis paper describes the development of CODEX in neuronal cell cultures derived from human iPSCs. ResultsWe differentiated human iPSCs into mixed neuronal and glial cultures on glass coverslips. We then developed and optimized a panel of 21 antibodies to phenotype iPSC-derived neuronal subtypes of cortical, dopaminergic, and striatal neurons, as well as astrocytes, and pre-and postsynaptic proteins. Comparison with existing methodsCompared to standard immunocytochemistry, CODEX oligoconjugated fluorophores circumvent antibody host interactions and allow for highly customized multiplexing. ConclusionWe show that CODEX can be applied to iPSC neuronal cultures and developed fixation and staining protocols for the neurons to sustain the multiple wash-stain cycles of the technology. Furthermore, we demonstrate both cellular and subcellular resolution imaging of multiplexed biomarkers in the same samples. CODEX is a powerful technique that complements other single-cell omics technologies for in-depth phenotype analysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/479039v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@68d49dorg.highwire.dtl.DTLVardef@5ea424org.highwire.dtl.DTLVardef@16a763forg.highwire.dtl.DTLVardef@7950fc_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOC_FLOATNO CODEX(R) Multiplex Imaging in human iPSC neurons [A-D] Schematic drawings of the tools and steps used for Co-Detection by indEXing (CODEX) imaging. [A] Target-specific antibodies are conjugated to unique DNA oligonucleotide barcodes. Fluorescent reporter (excitation wavelength at 488 nm, 550 nm, or 647 nm/Cy5) carrying the complementary DNA (to the barcode) enables barcode-specific binding of the reporter to the antibody and detection by fluorescence microscopy. [B] Neuronal cell cultures are prepared for the CODEX staining and imaging by several fixation steps with different PFA concentrations followed by incubation with 100% acetone. Residual acetone is removed by drying the sample. After rehydration with PBS, autofluorescence is quenched by exposure to broad-spectrum LED light. Following a pre-staining fixation step, the sample is incubated with a mix of all conjugated primary antibodies. Excessive, unbound antibodies are removed by a washing step, leaving only the bound antibodies followed by a final post-staining fixation. [C] The CODEX Instrument Manager performs the multicycle run and controls the microscope software for automated addition of reporters, imaging, and washing of the samples (pre-stained with primary antibodies) to remove reporters from each cycle. After imaging, bound reporters are removed without damaging the tissue using a solvent, and the next set of reporters (conjugated to different barcodes) are added. [D] CODEX(R) Processor processes raw files and performs stitching, deconvolution, background subtraction, and cell segmentation. The processed images can be viewed and analyzed with the CODEX(R) Multiplex Analysis Viewer (MAV) plugin using Fiji software. C_FIG

neuroscience↗

bioRxiv2019

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform specific start-loss mutations of essential genes can cause genetic diseases

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early onset, therapy resistant seizures and developmental delay. Here we report on 12 individuals from 10 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly and visual disturbance. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A>G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in brain cell types, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modelled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE. Importantly, it also shows that isoform specific start-loss mutations causing expression loss of a tissue relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

genetics↗

bioRxiv2019

Aceclofenac Fast Dispersible Tablet Formulations: Effect of different concentration levels of Avicel PH102 on the compactional, mechanical and drug release characteristics

Objective of this study was based on the formulation development of fast dispersible Aceclofenac tablets (100mg) and to evaluate the influence of pharmaceutical mixtures of directly compressible Avicel PH102 with Mannitol and Acdisol on the compressional, mechanical characteristics and drug release properties. Fifteen different aceclofenac formulations were developed by central composite rotatable design (CCRD). Among them best possible formulations (FA-FH) were selected on the basis of micromeritic properties, appropriate tablet weight and disintegration time for further study. Tablets were compressed by direct compression method using hand held hydraulic press with a compressional force ranging from 8 to 80 MN/m2 (MPa). Pre and post compression studies were performed and the compressed formulations (FA-FH) were assessed for different quality tests. The Heckel and Kawakita equations were applied for determination of compressional behavior of formulations. The quality attributes suggested that formulation (FB) containing avicel PH 102 (20%), mannitol (25%) and ac-di-sol (3%) as best optimized formulation showing better mechanical strength i.e. hardness 37.75 {+/-} 0.14N, tensile strength 5.67MN/m2 and friability 0.34%. Furthermore, compressional analysis of FB showed lowest PY value 59.52 MN/m2 and Pk value 1.040 MN/m indicating plasticity of the material. Formulation FB disintegrated rapidly within 21 seconds and released 99.92 % drug after 45min in phosphate buffer pH 6.8. Results of drug release kinetics showed that formulations FA-FH followed Weibull and First-order models in three different dissolution media. Avicel based formulation mixture exhibit excellent compactional strength with rapid disintegration and drug release.

pharmacology and toxicology↗

bioRxiv2019

Cardiomyocyte cell cycling, maturation, and growth by multinucleation in postnatal swine

AimsCardiomyocyte (CM) cell cycle arrest, decline of mononucleated-diploid CMs, sarcomeric maturation, and extracellular matrix remodeling are implicated in loss of cardiac regenerative potential in mice after birth. Recent studies show a 3-day neonatal regenerative capacity in pig hearts similar to mice, but postnatal pig CM growth dynamics are unknown. We examined cardiac maturation in postnatal pigs and mice, to determine the relative timing of developmental events underlying heart growth and regenerative potential in large and small mammals.\n\nMethods and ResultsLeft ventricular tissue from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed to span birth, weaning, and adolescence in pigs, compared to similar physiological timepoints in mice. Collagen remodeling increases by P7 in postnatal pigs, but sarcomeric and gap junctional maturation only occur at 2mo. Also, there is no postnatal transition to beta-oxidation metabolism in pig hearts. Mononucleated CMs, predominant at birth, persist to 2mo in swine, with over 50% incidence of mononucleated-diploid CMs at P7-P15. Extensive multinucleation with 4-16 nuclei per CM occurs beyond P30. Pigs also exhibit increased CM length relative to multinucleation, preceding increase in CM width at 2mo-6mo. Further, robust CM mitotic nuclear pHH3 activity and cardiac cell cycle gene expression is apparent in pig left ventricles up to 2mo. By contrast, in mice, these maturational events occur concurrently in the first two postnatal weeks alongside loss of cardiac regenerative capacity.\n\nConclusionsCardiac maturation occurs over a 6mo postnatal period in pigs, despite a similar early-neonatal heart regenerative window as mice. Postnatal pig CM growth includes increase in CM length alongside multinucleation, with CM cell cycle arrest and loss of mononucleated-diploid CMs occurring at 2mo-6mo. These CM characteristics are important to consider for pig preclinical studies and may offer opportunities to study aspects of heart regeneration unavailable in other models.

developmental biology↗

bioRxiv2019

SCAR FORMATION AND DECREASED CARDIAC FUNCTION FOLLOWING ISCHEMIA/REPERFUSION INJURY IN 1-MONTH-OLD SWINE

Studies in mice show a brief neonatal period of cardiac regeneration with minimal scar formation. Less is known about reparative mechanisms in large mammals. A transient cardiac injury approach (ischemia/reperfusion, IR) was used in weaned postnatal day (P)30 pigs, to assess regenerative repair in young large mammals. Female and male P30 pigs were subjected to cardiac ischemia (1 hour) by occlusion of the left anterior descending artery followed by reperfusion, or to sham operation. Following IR, myocardial damage occurred, with cardiac ejection fraction significantly decreased 2 hours post-ischemia. No improvement or worsening of cardiac function to the 4 week study end-point was observed. Histology demonstrated cardiomyocyte (CM) cell cycling at 2-months-of-age in multinucleated CMs in both sham-operated and IR pigs. Regional scar formation and inflammation in the epicardial region proximal to injury were observed 4 weeks post-IR. Sex differences were found, suggestive of females creating a greater fibrotic response with worse cardiac function, highlighting the importance of representing both sexes in cardiac injury studies. Together, our results describe an effective novel cardiac injury model in P30 swine, at a time when CMs are still cycling. Pigs subjected to IR show myocardial damage with a prolonged decrease in cardiac function, formation of a small, regional scar with increased inflammation. These data demonstrate that P30 pigs do not regenerate myocardium, even in the presence of CM mitotic activity, but form a scar after transient IR injury.\n\nNEW & NOTEWORTHYHere, we report for the first time ischemia/reperfusion (IR) cardiac injury in 1-month-old (P30) pigs. This model of IR injury highlights lack of cardiac regeneration, even in the presence of cardiomyocyte (CM) cell cycling in young swine. An effective injury approach is described for use in large mammals to investigate cardiac function, CM cell cycling, extracellular matrix (ECM) remodeling, and gene expression changes, while highlighting the importance of studying both sexes.

developmental biology↗