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Several emerging therapies kill cancer cells primarily by inducing necrosis. As necrosis activates immune cells, potentially, uncovering the molecular drivers of anticancer therapy-induced necrosis could reveal approaches for enhancing immunotherapy efficacy. To identify necrosis-associated genes, we performed a genome-wide CRISPR-Cas9 screen with negative selection against necrosis-inducing preclinical agents BHPI and conducted follow-on experiments with ErSO. The screen identified transient receptor potential melastatin member 4 (TRPM4), a calcium-activated, ATP-inhibited, sodium-selective plasma membrane channel. Cancer cells selected for resistance to BHPI and ErSO exhibited robust TRPM4 downregulation, and TRPM4 reexpression restored sensitivity to ErSO. Notably, TRPM4 knockout (TKO) abolished ErSO-induced regression of breast tumors in mice. Supporting a broad role for TRPM4 in necrosis, knockout of TRPM4 reversed cell death induced by four additional diverse necrosis-inducing cancer therapies. ErSO induced anticipatory unfolded protein response (a-UPR) hyperactivation, long-term necrotic cell death, and release of damage-associated molecular patterns that activated macrophages and increased monocyte migration, all of which was abolished by TKO. Furthermore, loss of TRPM4 suppressed the ErSO-induced increase in cell volume and depletion of ATP. These data suggest that ErSO triggers initial activation of the a-UPR but that it is TRPM4-mediated sodium influx and cell swelling, resulting in osmotic stress, which sustains and propagates lethal a-UPR hyperactivation. Thus, TRPM4 plays a pivotal role in sustaining lethal a-UPR hyperactivation that mediates the anticancer activity of diverse necrosis-inducing therapies. SIGNIFICANCE: A genome-wide CRISPR screen reveals a pivotal role for TRPM4 in cell death and immune activation following treatment with diverse necrosis-inducing anticancer therapies, which could facilitate development of necrosis-based cancer immunotherapies.
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Adenosina Trifosfato , Canales Catiónicos TRPM , Ratones , Animales , Necrosis/metabolismo , Muerte Celular , Membrana Celular/metabolismo , Adenosina Trifosfato/metabolismo , Sodio/metabolismo , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
INTRODUCTION: The CGG repeats in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) gene shows increased instability upon maternal transmission. Maternal FMR1 intermediate (45-54 repeats) and premutation (PM: 55-<200 repeats) alleles usually expand to full mutation (>200 repeats) alleles in offspring and consequently, cause fragile X syndrome (FXS) in them. METHODS: In a prospective cohort study, Pakistani pregnant women in prenatal care were first screened for FMR1 expanded alleles. In the follow-up, pregnancy outcomes in women carrying FMR1 expanded alleles were recorded and their newborn offspring were also screened for FXS. RESULTS: In a total of 1950 pregnant women, 89 (4.6%) were detected carriers for FMR1 expanded alleles; however, rates of detection of expanded alleles were found significantly high in women with a history of FXS. In addition, miscarriages and birth of affected newborns with FXS were significantly more common in women carrying large size PM alleles and had a history of FXS (P = 0.0494 and P = 0.0494, respectively). CONCLUSIONS: The current study provides the first evidence of screening Pakistani pregnant women for FMR1 expanded alleles in prenatal care. Moreover, the miscarriage was also detected as a clinical predictor for FXS. IMPACT: Offspring would have a higher risk of developing FXS due to maternal FMR1 alleles expansions during transmission. This is the first prospective cohort study in Pakistan for finding FMR1 allelic status of pregnant women and their newborn offspring in follow-up. The robust offspring risk for FXS estimated in this study may be valuable information for genetic counseling of women carriers for FMR1 expanded alleles. The family history and miscarriage were detected as effective indicators for FXS carrier screening in Pakistani women.
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Aborto Espontáneo , Síndrome del Cromosoma X Frágil , Humanos , Femenino , Recién Nacido , Embarazo , Alelos , Estudios Prospectivos , Aborto Espontáneo/genética , Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/genética , Mutación , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genéticaRESUMEN
Background: Dystrophic Epidermolysis bullosa (DEB) is a rare, severe subtype of epidermolysis bullosa (EB), characterized by blisters and miliary rashes of the skin. Dystrophic EB (DEB) includes variants inherited both in an autosomal-dominant or autosomal-recessive manner. Recessive dystrophic EB (RDEB) is divided into many subtypes and prevails as a result of biallelic genetic mutations in COL7A1 gene encoding type VII collagen, a major stabilizing molecule of the dermo-epidermal junction. The blister formation is mainly due to the variable structural and functional impairment of anchoring fibrils in VII collagen (COLVII), responsible for the adhesion of the epidermis to the dermis. Method: Three Pakistani families (A, B and C) affected with congenital dystrophic epidermolysis bullosa were recruited in the present study. The whole-exome sequencing (WES) approach was utilized for the detection of the pathogenic sequence variants in probands. The segregation of these variants in other participants was confirmed by Sanger sequencing. Results: This study identified a novel missense variant c.7034G>A, p. Gly2345Asp in exon 91, a novel Frameshift mutation c.385del (p. His129MetfsTer18) in a homozygous form in exon no 3, and a previously known nonsense variation (c.1573 C>T; p. Arg525Ter) in exon 12 of COL7A1 gene in families A, B, and C, respectively, as causative mutations responsible for dystrophic epidermolysis bullosa in these families. Conclusion: Our study validates the involvement of the COL7A1 gene in the etiology of dystrophic epidermolysis bullosa. It further expands the COL7A1 gene mutation database and provides an additional scientific basis for diagnosis, genetic counseling, and prognosis purposes for EB patients.
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PURPOSE: Women of reproductive age who carry fragile X premutation (PM) alleles have 56 to 200 CGG repeats in the 5'-untranslated region of FMR1 gene are at increased risk for producing children with intellectual disabilities (ID) or autism spectrum disorders (ASD) due to expansion of PM alleles to full mutation alleles (> 200 repeats) during maternal transmission. METHODS: In present study fragile X PM carrier screening was performed in total 808 women who were consulting primary health care centers for preconception care in Khyber Pakhtunkhwa region of Pakistan between April, 2018 and December, 2020. Polymerase chain reaction (PCR) was performed for detection of PM carrier women and the CGG repeats number was confirmed by Southern blotting and capillary electrophoresis. RESULTS: The prevalence rate for PM carriers among preconception women was found to be 0.7% that was contributed by 0.5% women in risk group (RG1) with family history of ID and 0.2% in risk group 2 (RG2) with family history of ASD. PM carrier women had at least one affected child or sibling. In addition, the preconception women with FMR1 PM alleles were found to be at increased risk for primary ovary insufficiency (RG1: P = 0.0265, RG2: P = 0.0389), postpartum depression (RG1: P = 0.0240, RG2: P = 0.0501) and neuropsychiatric disorders (RG1: P = 0.0389, RG2: P = 0.0432). CONCLUSIONS: Current study provides first evidence of fragile X PM carrier screening in Pakistani preconception women in primary care consultation. Findings of current study may help to improve preconception care and to reduce burden of fragile X associated disorders in our population.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Niño , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/diagnóstico , Síndrome del Cromosoma X Frágil/epidemiología , Síndrome del Cromosoma X Frágil/genética , Humanos , Masculino , Mutación , Pakistán , Atención Primaria de Salud , Derivación y ConsultaRESUMEN
The silver nanoparticles (AgNPs) were synthesized via green synthesis approach using Euporbia serpens Kunth aqueous extract. The synthesized AgNPs were characterized by UV-visible spectroscopy and Furrier Transformer Infra-Red spectroscopy to justify the reduction and stabilization of AgNPs from its precursors. AgNPs characteristic absorption peak was observed at 420 nm in the UV-visible spectrum. The SEM and TEM analysis demonstrated the spherical shape of the synthesized nanoparticles with particle sizes ranging from 30 nm to 80 nm. FTIR transmission bands at 2920 cm-1, 1639 cm-1, 1410 cm-1, 3290 cm-1, and 1085 cm-1 were attributed to C-H, C=O, C-C, N-H, and C-N functional groups, respectively. XRD peaks could be attributed to (111), (200), (220), and (311) crystalline plane of the faced-centered cube (FCC) crystalline structure of the metallic silver nanoparticles. The AgNPs showed good antibacterial activity against all the tested bacteria at each concentration. The particles were found to be more active against Escherichia coli (E. coli) with 20 ± 06 mm and Salmonella typhi (S. typhi) with 18 ± 0.5 mm zone of inhibition in reference to standard antibiotic amoxicillin with 23 ± 0.3 mm and 20 ± 0.4 mm zone of inhibition, respectively. Moderate antifungal activities were observed against Candida albicans (C. albicans) and Alternaria alternata (A. alternata) with zone of inhibitions 16.5 mm and 15 mm, respectively, compared to the standard with 23 mm of inhibition. Insignificant antifungal inhibition of 7.5 mm was observed against Fusarium gramium (F. gramium). All the tested concentrations of AgNPs showed comparable % RSA with the standard reference ascorbic acid in the range sixty percent to seventy five percent. The percent motility at 3 hours postincubation showed quick response and most Tetramorium caespitum were found deceased or paralyzed. Similarly, the percent mortality showed a linear response at concentration and time. It was observed that 1 µg/mL to 2 µg/mL concentration of AgNPs displayed a significant cytotoxic activity against Artemia salina with LD50 of 5.37 and 5.82, respectively.
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Antiinfecciosos , Antioxidantes , Citotoxinas , Euphorbia/química , Nanopartículas del Metal , Extractos Vegetales/química , Plata , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antioxidantes/síntesis química , Antioxidantes/química , Antioxidantes/farmacología , Citotoxinas/síntesis química , Citotoxinas/química , Citotoxinas/farmacología , Nanopartículas del Metal/química , Nanopartículas del Metal/uso terapéutico , Pruebas de Sensibilidad Microbiana , Plata/química , Plata/farmacologíaRESUMEN
We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.
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Materiales Biocompatibles/farmacocinética , Carbono/farmacocinética , Reactivos de Enlaces Cruzados/farmacocinética , Colorantes Fluorescentes/farmacocinética , Luminiscencia , Puntos Cuánticos/química , Materiales Biocompatibles/química , Carbono/química , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/química , Teoría Funcional de la Densidad , Colorantes Fluorescentes/química , Humanos , Ensayo de Materiales , Estructura Molecular , Imagen Óptica , Procesos Fotoquímicos , Espectroscopía de Fotoelectrones , Propiedades de SuperficieRESUMEN
Per- and polyfluoroalkyl substances (PFAS) are a group of human-made compounds with strong C-F bonds, and have been used in various manufacturing industries for decades. PFAS have been reported to deleterious effect on human health, which has led to studies identifying the possible toxicity and toxicity routes of these compounds. We report that these compounds have the potential to cause epigenetic modifications, and to induce dysregulation in the cell proliferation cycle as well as apoptosis in A549 lung cancer cells when exposed to 10-, 200- and 400 µM concentrations of each compound. Our studies show that exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) may cause hypomethylation in the epigenome, but changes in the epigenetic makeup are not evident upon exposure to GenX. We establish that exposure to lower doses of these compounds causes the cells' balance to shift to cell proliferation, whereas exposure to higher concentrations shifts the balance more towards apoptosis. Furthermore, the apoptosis pathway upon exposure to GenX, PFOA, and PFOS has also been identified. Our findings suggest that exposure to any of these compounds may have profound effects in patients with pre-existing lung conditions or could trigger lung cancinogenesis.
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OBJECTIVE: To evaluate the knowledge, attitude and practice of self-medication in medical undergraduate students. METHODS: This cross-sectional questionnaire-based survey was conducted from January to June, 2017, among undergraduate students of Ayub Teaching Hospital, Women Medical College, International Medical College and Frontier Medical College in Abbottabad, Pakistan. The sample comprised students of first, second, third, fourth and fifth professional year. Data was collected using self-generated questionnaire. RESULTS: Of the 400 subjects approached, 300(75%) returned the questionnaire fully completed. Of them, 208 (69.3%) were females and 92(30.66%) were males. The most common age group was 22-25 years 182(60.7%). Self-medication was found to be prevalent among 297(99%) subjects and 139(46.3%) respondents had practised self-medication in the preceding 6 months more than two times. Over-the-counter drugs were commonly used for the self-medication in 295(98.3%) cases. Overall, 285(95%) respondents had a positive attitude towards self-medication. Media was found to be the most common source of information for 93(31%) cases. CONCLUSIONS: High prevalence of self-medication was noticed, with over-the-counter drugs being the most commonly used. Proper educational awareness programmes about self-medication can control the issue.
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Conocimientos, Actitudes y Práctica en Salud , Automedicación , Estudiantes de Medicina , Adulto , Actitud Frente a la Salud , Estudios Transversales , Femenino , Humanos , Masculino , Medicamentos sin Prescripción/uso terapéutico , Pakistán/epidemiología , Prevalencia , Automedicación/métodos , Automedicación/psicología , Automedicación/estadística & datos numéricos , Estudiantes de Medicina/psicología , Estudiantes de Medicina/estadística & datos numéricos , UniversidadesRESUMEN
Regions of Iran, Afghanistan, Pakistan and northwestern India have been proposed as the place of origin of Mus musculus castaneus. But despite the fact that Pakistan encompasses an important part of its range, M. m. castaneus populations in Pakistan have not been the subject of intensive genetic and biogeographic studies, except for a very small number of samples included in past studies. We studied genetic variation in M. m. castaneus (CAS) from northern Punjab Province, Pakistan, by using cytochrome b (Cytb) analysis in a sample of 98 individuals. Median-joining network revealed four well differentiated CAS sub-lineages coexisting within a small geographical region; these had previously been thought to have largely non-overlapping geographic distributions. Moreover, haplotypes from Pakistan occupied a central position in the network and all identified global haplotypes were also present in Pakistan. All identified CAS sub-lineages proved to be highly diverse on the basis of haplotype and nucleotide diversity indices. Tajima's D test and Fu's Fs tests of neutrality suggest recent population expansions in all sub-lineages. Expansion times were estimated as 21,760-134,930, 10,800-64,400 and 4950-30,665 ybp using substitution rates of 2.5%, 5% and 11%, respectively. Our results support the hypothesis that northern Punjab Province in Pakistan is the most likely source area for M. m. castaneus, and that the CAS sub-lineages in this region have undergone rapid population expansion events at different time periods, which appear to have benefitted from human-mediated transport, although one of them clearly predates the establishment of human settlements in this region.
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ADN Mitocondrial/genética , Variación Genética , Ratones/genética , Animales , Haplotipos , Pakistán , Filogenia , FilogeografíaRESUMEN
OBJECTIVE: To investigate the efficacy and impact of Protection Against Harassment of Women at Workplace Act 2010 in the public health sector in its socio-legal perspective. METHODS: This cross-sectional study was conducted from July to December 2014 in Abbottabad, Pakistan, and comprised subjects selected from 53 basic health units managed by the government. SPSS 20 was used for statistical analysis. RESULTS: Of the 450 questionnaires, 430(96.6%) were returned duly filled. Overall, 40% male (120 respondents) and 26% female (34 respondents) knew about the Act. Besides, 39% males (117 respondents) and 63% females (82 respondents) appeared unsatisfied with the complaint mechanism prescribed in the Act; all the respondents established that no case had been registered after the introduction of the Act. Overall, 31% males (93 respondents) and 57% females (74 respondents) thought that without the health governance commitment and social support structure the Act could not work. CONCLUSIONS: The Protection Against Harassment of Women at Workplace Act 2010 was considered ineffective by the respondents who were concerned about the lack of social support system for the Act.