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Khan, A.

Publications and source records attributed to Khan, A..

At least 145 records · Page 8Linked to original sources

Mapping the subcortical connectome using in vivo diffusion MRI: feasability and reliability

Tractography combined with regions of interest (ROIs) has been used to non-invasively study the structural connectivity of the cortex as well as to assess the reliability of these connections. However, the subcortical connectome (subcortex to subcortex) has not been comprehensively examined, in part due to the difficulty of performing tractography in this complex and compact region. In this study, we performed an in vivo investigation using tractography to assess the feasibility and reliability of mapping known connections between structures of the subcortex using the test-retest dataset from the Human Connectome Project (HCP). We further validated our observations using a separate unrelated subjects dataset from the HCP. Quantitative assessment was performed by computing tract densities and spatial overlap of identified connections between subcortical ROIs. Further, known connections between structures of the basal ganglia and thalamus were identified and visually inspected, comparing tractography reconstructed trajectories with descriptions from tract-tracing studies. Our observations demonstrate both the feasibility and reliability of using a data-driven tractography-based approach to map the subcortical connectome in vivo.

neuroscience↗

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association between TOP1MT and autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed in TOP1MT knockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

cell biology↗

Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila Melanogaster (Pupal Retina)

A novel pair of protein Tyrosine Phosphatases in Drosophila Melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized having fundamental role in cellular processes including effects on metabolism, cell proliferation and differentiation. This study presents comparative homology modeling of low molecular weight phosphotyrosine protein phosphatase (PTPs) from Drosophila melanogaster (Dr-PTPs) and their complexation with potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homology is approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel {beta} sheet with flanking helices on both sides, showing two right-handed {beta}--{beta} motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs activation. The structure guided studies of both proteins clearly reveal a common mechanism of action, inhibitor binding at the active site and will expected to contribute towards the basic understanding of functional association of this enzyme with other molecules.

biochemistry↗

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↗

Organ Scaffold Characterizations Using Intravital Microscopy

This study contains intravital microscopy (IVM) data to characterize organ scaffolds created using decellularization. Decellularization creates cell-free collagen-based matrices from native organs, which can be used as scaffolds for regenerative medicine applications. This data set contains in vivo assays evaluating the effectiveness of decellularization and structural and functional integrity of the acellular nephron in the post-transplantation environment. Qualitative and quantitative assessments of scaffold DNA concentrations, tissue fluorescence signals, structural and functional integrities of various decellularized nephron segments, and velocity within the microcirculation were acquired and compared to the native (non-transplanted) organ. Cohorts of 2-3-month-old male Sprague Dawley rats were used: non-transplanted (n = 4), transplanted day 0 (n=4), transplanted day 1 (n = 4), transplanted day 2 (n = 4), and transplanted day 7 (n = 4). OIF formatted files illustrate IVM measurement processes and are made publicly available in a data repository. Such data is intended to support scientific reproducibility, complement future renal studies, and extend the use of this powerful imaging application to analyze other organ scaffold systems. O_TBL View this table: org.highwire.dtl.DTLVardef@4937b8org.highwire.dtl.DTLVardef@e91b6forg.highwire.dtl.DTLVardef@83eeforg.highwire.dtl.DTLVardef@15be0d4org.highwire.dtl.DTLVardef@105a2e5_HPS_FORMAT_FIGEXP M_TBL C_TBL Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.18703949.

bioengineering↗

mTORC2 Drives ZFX-mediated Ganglioside Biosynthesis to Promote Breast Cancer Progression.

Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside biosynthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in luminal breast cancer-specific MCF-7 and BT-474 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian luminal breast cancer tissues, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.

cancer biology↗

Integrated cyto-physiological and proteomic analyses reveal new insight into CMS mechanism in a novel upland cotton CMS line LD6A

Cytoplasmic male sterile (CMS) system has extensively been exploited for hybrid vigor in plant breeding programs. However, its application in many crops is limited due to poor understanding of molecular mechanism of fertility restoration. Using advanced analytical approaches, we elucidated molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed for physiological and proteomic changes during the development process. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in CMS and its maintainer line. Transmission electron micrographs of anther tapetum showed that inner ridge of CMS mitochondria was relatively indistinct than that of LD6B with narrower membranous space at tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.

developmental biology↗

A myosin chaperone, UNC-45A, is a novel regulator of intestinal epithelial barrier integrity and repair

The actomyosin cytoskeleton serves as a key regulator of the integrity and remodeling of epithelial barriers by controlling assembly and functions of intercellular junctions and cell-matrix adhesions. While biochemical mechanisms that regulate activity of non-muscle myosin II (NM-II) in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. UNC-45A is a cytoskeletal chaperone that is essential for proper folding of NM II heavy chains and myofilament assembly. We found abundant expression of UNC-45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC-45A were decreased in colonic epithelium of patients with ulcerative colitis. CRISPR/Cas9-mediated knock-out of UNC-45A in HT-29 and SK-CO15 IEC disrupted epithelial barrier integrity, impaired assembly of epithelial adherence and tight junctions and attenuated cell migration. Consistently, decreased UNC-45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti-migratory effects of UNC-45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC-45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC-45A in controlling IEC junctions and motility. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.

cell biology↗

PRMT5 in T cells drives Th17 responses, mixed granulocytic inflammation and severe allergic airway inflammation

Severe asthma is characterized by steroid insensitivity and poor symptom control, and is responsible for the majority of asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding in mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in asthmatic lungs. Here, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, ROR-{gamma}t and Th17 responses, with PRMT5-dependent increases in ROR-{gamma}ts agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

immunology↗

CD20+ T cells are associated with inflammatory responses in experimental arthritis.

CD20+ T cells comprise a small but highly inflammatory subset that has been implicated in autoimmunity, including rheumatoid arthritis (RA). We sought to characterise the CD20+ T cell subset at the site of inflammation in murine collagen-induced arthritis (CIA) model of RA and investigate the phenotype and functional relevance of CD3+CD20+ T cells in the lymph nodes and arthritic joints using flow cytometry and immunohistochemistry. We demonstrate that CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells are expanded in the draining lymph nodes of CIA mice. In addition, compared to naive mice and those that did not develop clinical symptoms, CD20 expressing T cells of arthritic mice produced increased levels of pro-inflammatory cytokines (GM-CSF, TNF-a, IL-17, and INF-g). Notably, CD3+CD4+CD20+ and CD3+CD8+CD20+ T cells of disease mice were enriched with CXCR5+PD-1+ T follicular helper cells and CXCR5-PD-1+ peripheral T helper cells, subsets of T cells that have been implicated in promoting B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues in RA. Importantly, CD3+CD20+ T cells were detected in the inflamed regions in the lymph nodes and paws of arthritic mice. Our findings suggest that CD20+ T cells are associated with inflammatory responses in the arthritic joint and may exacerbate pathology by promoting inflammatory B cell responses.

immunology↗

Multi-modal profiling of the extracellular matrix of human fallopian tubes and serous tubal Intraepithelial carcinomas

Recent evidence supports the fimbriae of the fallopian tube as a potential origin site for high-grade serous ovarian cancer (HGSOC). The progression of many solid tumors is accompanied by changes in the microenvironment, including alterations of the extracellular matrix (ECM). The ECM of fallopian tube and HGSOC has not been well characterized. Therefore, we sought to determine the ECM composition of the benign fallopian tube and how it changes with the onset of serous intraepithelial carcinomas (STICs), precursor of HGSOC. The ECM composition of benign human fallopian tube was first defined from a meta-analysis of published proteomic datasets and identified 190 ECM proteins. We then conducted de novo proteomics using ECM enrichment and identified 88 proteins, 7 of which were not identified in prior studies. We further investigated the levels and localization of seven of these ECM proteins (type I, III, and IV collagens, fibronectin, laminin, versican, perlecan) and hyaluronic acid using multi-spectral immunohistochemical staining of fimbriae from patients with benign conditions or STICs. Quantification revealed an increase in stromal fibronectin and a decrease in epithelial versican in STICs. Our results provide an in-depth picture of the ECM in the benign fallopian tube and identified ECM changes that accompany STIC formation.

cancer biology↗

Myelin imaging of the basal forebrain in first-episode psychosis

Cholinergic dysfunction has been implicated in the pathophysiology of psychiatric disorders such as schizophrenia, depression, and bipolar disorder. The basal forebrain (BF) cholinergic nuclei, defined as cholinergic cell groups Ch1-3 and Ch4 (Nucleus Basalis of Meynert; NBM), provide extensive cholinergic projections to the rest of the brain. Here, we examined microstructural neuroimaging measures of the cholinergic nuclei in patients with untreated psychosis ([~] 31 weeks of psychosis, <2 defined daily dose of antipsychotics) and used Magnetic Resonance Spectroscopy (1H-MRS) and transcriptomic data to support our findings. We used a cytoarchitectonic atlas of the BF to map the nuclei and obtained measures of myelin (quantitative T1, or qT1 as myelin surrogate) and microstructure (axial diffusion; AxD). In a clinical sample (n=85; 29 healthy controls, 56 first-episode psychosis), we found significant correlations between qT1 and 1H-MRS-based dorsal anterior cingulate choline in healthy controls, while this relationship was disrupted in FEP. Case-control differences in qT1 and AxD were observed in the Ch1-3, with increased qT1 (reflecting reduced myelin content) and AxD (reflecting reduced axonal integrity). We found clinical correlates between left NBM qT1 with manic symptom severity, and AxD with negative symptom burden in FEP. Intracortical and subcortical myelin maps were derived and correlated with BF myelin. BF-cortical and BF-subcortical myelin correlations demonstrate known projection patterns from the BF. Using data from the Allen Human Brain Atlas, cholinergic nuclei showed significant enrichment for schizophrenia and depression-related genes. Cell-type specific enrichment indicated enrichment for cholinergic neuron markers as expected. Further relating the neuroimaging correlations to transcriptomics demonstrated links with cholinergic receptor genes and cell type markers of oligodendrocytes and cholinergic neurons, providing biological validity to the measures. These results provide genetic, neuroimaging, and clinical evidence for cholinergic dysfunction in schizophrenia and other psychiatric disorders such as depression.

neuroscience↗

Sugar Beet Extract Acts as a Natural Bio-Stimulant for Physio-Biochemical analysis of Hordeum Vulgare L. under Induced Salinity Stress

Change in climate of the entire globe due to elevated temperature and minimum annual rainfall in barren zone frequently leads to salinity of soil. The current study was aimed to evaluate the importance of sugar beet extract (SBE) as a bio-stimulant to improve the adverse damages of induced salinity stress (40mM) on growth, oosmolytes and antioxidant defense system of barley (Hordeum vulgare L.). Pot experiment was carried in green house under different concentrations of SBE (10%, 20%, 30%, 40%, 50%) pre-soaked seeds of Hordeum vulgare for 5 hours SBE was analyzed for glycine betaine (100mmol/kg), betalains (1.3mg/l), phenolics (1.30g/100ml), flavonoids (0.59mg/ml), carotenoids (0.23ml/100ml), vitamin E (0.002%), vitamin C (8.04g/100ml), sugar (8g/100ml), protein (1.39mg/100ml), and oxalic acid (38mg/100ml) while Ca (13.72mg/l), Mg (7.121 mg/l) and K (11.45mg/l) contents were also determined. We found significant improvement in germination parameters of Hordeum vulgare L. via SB extract on coefficient of velocity of emergence (CVE), mean emergence time (MET), germination energy (GE), timson germination index (TGI), germination rate index (GRI) and time to 50% emergence (E50) under induced salinity stress. However, photosynthetic pigments, e.g., chlorophyll and carotenoids were enhanced using 40% SB extract, soluble sugar, protein, proline, POD, MDA with 50% SB extract while SOD and H2O2 in 20% SBE, respectively. Our findings suggested that SB extract promotes both agronomical and physiological attributes, is a positive way to enhance our economy by increasing crop yields in arid and semi-arid areas along with plant tolerance to under induced salinity stress. HighlightsThe recent study is of utmost importance owing to the following reasons:[tpltrtarr] Changes in climatic condition results in salinity, droughts, floods, earthquakes, fluctuation in temperature and other environmental hazards. [tpltrtarr]Salinity stress has myriad of damaging effects on crop growth and productivity and becoming a global issue of major concern especially in the Asian countries including Pakistan. Statistical analysis showed that 6.30 million of lands is affected with salinity. [tpltrtarr]Barley (Hordeum Vulgare L.) being an important crop of economic and nutritional value has been facing salinity stress condition in Pakistan, resulting in productivity reduction and ultimately paving the way for food scarcity and crippling economy. [tpltrtarr]The sole purpose of this research article is to assess and ameliorate the physio-biochemical responses via application of sugar beet extract as bio-stimulants in Barley (Hordeum Vulgare L.) grown under induced salinity stress; in order to cope with the dire consequences of salinity stress especially in the southern areas of Pakistan being badly affected by salt stress. [tpltrtarr]Furthermore, there is a great need for adaptive measures suggested by the present research and other findings made by researchers in pursuit of tackling this alarming global menace.

plant biology↗

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis

The integral membrane protein Apq12 is an important nuclear envelope (NE)/ER modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here we identified a short amphipathic -helix (AH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AH is disrupted show NPC biogenesis and NE integrity defects, without impacting upon Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic -helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE during NPC biogenesis.

cell biology↗

Learning shapes cortical dynamics to enhance integration of relevant sensory input

Adaptive sensory behavior is thought to depend on processing in recurrent cortical circuits, but how dynamics in these circuits shapes the integration and transmission of sensory information is not well understood. Here, we study neural coding in recurrently connected networks of neurons driven by sensory input. We show analytically how information available in the network output varies with the alignment between feedforward input and the integrating modes of the circuit dynamics. In light of this theory, we analyzed neural population activity in the visual cortex of mice that learned to discriminate visual features. We found that over learning, slow patterns of network dynamics realigned to better integrate input relevant to the discrimination task. This realignment of network dynamics could be explained by changes in excitatory-inhibitory connectivity amongst neurons tuned to relevant features. These results suggest that learning tunes the temporal dynamics of cortical circuits to optimally integrate relevant sensory input. HighlightsO_LIA new theoretical principle links recurrent circuit dynamics to optimal sensory coding C_LIO_LIPredicts that high-SNR input dimensions activate slowly decaying modes of dynamics C_LIO_LIPopulation dynamics in primary visual cortex realign during learning as predicted C_LIO_LIStimulus-specific changes in E-I connectivity in recurrent circuits explain realignment C_LI

neuroscience↗

Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation, and that is involved in mitochondrial replication, transcription and translation. Two variants predicted to affect TOP1MT function (V1 - R198C and V2 - V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants had been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in knockout cells. Consistent with these TOP1MT variants contributing to the patient phenotype, comprehensive characterization suggests that both variants had impaired activity. Critically, neither variant was able to restore steady state levels of mitochondrial-encoded proteins, nor reduced oxidative phosphorylation when re-expressed in TOP1MT knockout cells. However, the two variants behaved differently in some respects. While the V1 variant was better at restoring transcript levels, the V2 variant was able to restore mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome, and how impairments in this important protein may lead to human pathology.

molecular biology↗

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

The genomic miscellany of malaria parasites can inform the intensity of transmission and identify potential deficiencies in malaria control programs. The aim of this study was to investigate the genomic miscellany, allele frequencies and multiplicity of infection (MOI) of P. falciparum. MethodsA total of 85 isolates from patients presenting to the local health centers with P. falciparum species were collected from 2017 to 2019. Parasite DNA was extracted from a total of 200 {micro}l whole blood per patient using the Qiagen DNA extraction kit according to manufactures instructions. The polymorphic region of msp-1, msp-2 and glurp loci were genotyped by using nested polymerase chain reactions followed by gel electrophoresis for fragment analysis. ResultsGenetic diversity and allelic frequencies of msp-1, msp-2 and glurp were identified in 85 blood samples. A total of 62 msp alleles were detected in which 30 for msp-1 and 32 for msp-2. For msp-1 the successful amplification occurred in (75/85) 88.23% isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp. For msp-1, the K1 allelic family was predominant at 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75) and 10.67% (8/75) respectively. In msp-2, the FC27 allelic family was the most abundant with 70.14% (47/67) compared to 3D7 with 67.16% (45/67). Nine glurp RII region genotypes were identified. The overall mean multiplicity of infection was 2.6 with1.8 and 1.4 for msp-1 and msp-2 respectively while for glurp RII genes (MOI=1.03). There was no significant association between multiplicity of infection and age group (Spearmans rank coefficient = 0.050; P = 0.6). There was significant correlation between MOI and parasite density for msp-2 allelic family. ConclusionOur study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates from malaria patients in Khyber Pakhtunkhwa Pakistan. In the present study the genotype data provided the valuable information which is essential for monitoring the impact of malaria eradication efforts in this region.

zoology↗

The Human Tumor Atlas Network (HTAN) Breast PreCancer Atlas: A multi-omic integrative analysis of ductal carcinoma in situ with clinical outcomes

Ductal carcinoma in situ (DCIS) is the most common precursor of invasive breast cancer (IBC), with variable propensity for progression. We have performed the first multiscale, integrated profiling of DCIS with clinical outcomes by analyzing 677 DCIS samples from 481 patients with 7.1 years median follow-up from the Translational Breast Cancer Research Consortium (TBCRC) 038 study and the Resource of Archival Breast Tissue (RAHBT) cohorts. We identified 812 genes associated with ipsilateral recurrence within 5 years from treatment and developed a classifier that was predictive of DCIS or IBC recurrence in both cohorts. Pathways associated with recurrence include proliferation, immune response, and metabolism. Distinct stromal expression patterns and immune cell compositions were identified. Our multiscale approach employed in situ methods to generate a spatially resolved atlas of breast precancers, where complementary modalities can be directly compared and correlated with conventional pathology findings, disease states, and clinical outcome. HIGHLIGHTS Development of a new classifier for DCIS recurrence or progression Outcome associated pathways identified across multiple data types and compartments Four stroma-specific signatures identified CNAs characterize DCIS subgroups associated with high risk invasive cancers

cancer biology↗