Mutational analysis and clinical investigations of medically diagnosed GSD 1a patients from Pakistan.

Glycogen storage disease type I (GSD I) is a rare autosomal recessive inborn error of carbohydrate metabolism caused by the defects of glucose-6-phosphatase complex (G6PC). Disease causing variants in the G6PC gene, located on chromosome 17q21 result in glycogen storage disease type Ia (GSD Ia). Age of onset of GSD Ia ranges from 0.5 to 25 years with presenting features including hemorrhage, hepatic, physical and blood related abnormalities. The overall goal of proposed study was clinical and genetic characterization of GSD Ia cases from Pakistani population. This study included forty GSD Ia cases presenting with heterogeneous clinical profile including hypoglycemia, hepatomegaly, lactic acidosis i.e., pH less than 7.2, hyperuricemia, seizures, epistaxis, hypertriglyceridemia (more than180 mg/dl) and sometimes short stature. All coding exons and intron-exon boundaries of G6PC gene were screened to identify pathogenic variant in 20 patients based on availability of DNA samples and willingness to participate in molecular analysis. Pathogenic variant analysis was done using PCR-Sanger sequencing method and pathogenic effect predictions for identified variants were carried out using PROVEAN, MutationTaster, Polyphen 2, HOPE, Varsome, CADD, DANN, SIFT and HSF software. Overall, 21 variants were detected including 8 novel disease causing variants i.e., G6PC (NM_000151.4):c.71A>C (p.Gln24Pro), c.109G>C(p.Ala37Pro), c.133G>C(p.Val45Leu), c.49_50insT c.205G>A(p.Asp69Asn), c.244C>A(p.Gln82Lys) c.322A>C(p.Thr108Pro) and c.322A>C(p.Cys284Tyr) in the screened regions of G6PC gene. Out of 13 identified polymorphisms, 3 were identified in heterozygous condition while 10 were found in homozygous condition. This study revealed clinical presentation of GSD Ia cases from Pakistan and identification of novel disease-causing sequence variants in coding region and intron-exon boundaries of G6PC gene.

Antimicrobial Dispensing Practices during COVID-19 and the Implications for Pakistan.

Antibiotics are one of the most frequently dispensed classes of medicines. However, excessive misuse and abuse enhances antimicrobial resistance (AMR). Previous studies in Pakistan have documented extensive dispensing of 'Watch' and 'Reserve' antibiotics, which is a concern. In view of this, there is a need to assess current dispensing patterns following COVID-19 in Pakistan. A cross-sectional study was undertaken, collecting dispensing data from 39 pharmacies and 53 drug stores from November 2022 to February 2023. Outlets were principally in urban areas (60.9%), with pharmacists/pharmacy technicians present in 32.6% of outlets. In total, 11,092 prescriptions were analyzed; 67.1% of patients were supplied at least one antimicrobial, 74.3% antibiotics, 10.2% antifungals and 7.9% anthelmintics. A total of 33.2% of antimicrobials were supplied without a prescription. Common indications for dispensed antibiotics were respiratory (34.3%) and gastrointestinal (16.8%) infections, which can be self-limiting. In addition, 12% of antibiotics were dispensed for the prevention or treatment of COVID-19. The most frequent antibiotics dispensed were ceftriaxone (18.4%) and amoxicillin (15.4%). Overall, 59.2% antibiotics were 'Watch' antibiotics, followed by 'Access' (40.3%) and 'Reserve' (0.5%) antibiotics. Of the total antibiotics dispensed for treating COVID-19, 68.3% were 'Watch' and 31.7% 'Access'. Overall, there appeared to be an appreciable number of antibiotics dispensed during the recent pandemic, including for patients with COVID-19, alongside generally extensive dispensing of 'Watch' antibiotics. This needs to be urgently addressed with appropriate programs among pharmacists/pharmacy technicians to reduce AMR.

Plant Growth-Promoting Rhizobacteria Eliminate the Effect of Drought Stress in Plants: A Review.

Plants evolve diverse mechanisms to eliminate the drastic effect of biotic and abiotic stresses. Drought is the most hazardous abiotic stress causing huge losses to crop yield worldwide. Osmotic stress decreases relative water and chlorophyll content and increases the accumulation of osmolytes, epicuticular wax content, antioxidant enzymatic activities, reactive oxygen species, secondary metabolites, membrane lipid peroxidation, and abscisic acid. Plant growth-promoting rhizobacteria (PGPR) eliminate the effect of drought stress by altering root morphology, regulating the stress-responsive genes, producing phytohormones, osmolytes, siderophores, volatile organic compounds, and exopolysaccharides, and improving the 1-aminocyclopropane-1-carboxylate deaminase activities. The use of PGPR is an alternative approach to traditional breeding and biotechnology for enhancing crop productivity. Hence, that can promote drought tolerance in important agricultural crops and could be used to minimize crop losses under limited water conditions. This review deals with recent progress on the use of PGPR to eliminate the harmful effects of drought stress in traditional agriculture crops.

Analysis of Wilson disease mutations in copper binding domain of ATP7B gene.

Wilson's disease (WD) is an autosomal recessive disorder, resulting from variations in ATP7B gene. Clinical heterogeneity, including neuropsychiatric and hepatic manifestations over a large range of age groups make diagnosis difficult. Most of WD patients suffer severe disabilities and even die. So, overall goal of proposed study is the genetic and clinical characterization of Wilson's disease cases from Pakistani population. Clinical data was collected, and patients were investigated for variations in selected ATP7B exons using PCR based Sanger sequencing. Pathogenic effect predictions for detected variants were carried out using PROVEAN, MutationTaster2, and HSF software's. Clinical heterogeneity was observed in patients including reduced serum ceruloplasmin, signs of chronic liver damage and raised 24 h urinary copper excretion. Mean age of onset was 11.3 years. Kayser-Fleischer rings were present in 75% of cases. About 82.5% patients belonged to inbred families. Patients having neurological disorder were above 12 years of age. Total ten variants in analyzed region of ATP7B gene, including a reported variation (p. L227Yfs*35) were found in patients. The study also identified 4 putative novel synonymous variants (c.251A>C, c.15T>A, c.6T>C, c.238C>T) and 5 reported polymorphisms (c.83C>A, c.39_40insCGGCG, p.V456L, c.39_40insCGCCG and c.1544-53A>C). Reliable understanding of clinical presentations and genotype-phenotype correlation provide insight to function and structure of ATP7B and may assist in disease prognosis and family counseling. The study revealed clinical presentation of Pakistani WD cases and identification of sequence variants in screened region of ATP7B.

Identification of Lignan Compounds as New 6-Phosphogluconate Dehydrogenase Inhibitors for Lung Cancer.

Targeting pentose phosphate pathway (PPP) enzymes has emerged as a promising strategy to combat cancer. 6-Phosphogluconate dehydrogenase (6-PGD), the third critical enzyme of the PPP, catalyzes oxidative decarboxylation of 6-phosphogluconate (6-PG) to produce ribulose-5-phosphate (Ru-5-P) and CO2. Overexpression of 6-PGD has been reported in multiple cancers and is recognized as a potential anticancer drug target. The current study is focused on the utilization of indispensable virtual screening tools for structure-based drug discovery. During the study, 17,000 natural compounds were screened against the 3-phosphoglycerate (3-PG) binding site of 6-PGD through a molecular operating environment (MOE), which revealed 115 inhibitors with higher selectivity and binding affinity. Out of the 115 best-fit compounds within the 6-PGD binding cavity, 15 compounds were selected and optimized through stringent in silico ADMET assessment models that justified the desirable pharmacokinetic, pharmacodynamic and physicochemical profiles of 5 ligands. Further protein−ligand stability assessment through molecular dynamics (MD) simulation illustrated three potential hits, secoisolariciresinol, syringaresinol and cleomiscosin A, with stable confirmation. Moreover, 6-PGD inhibitor validation was performed by an in vitro enzymatic assay using human erythrocytes purified 6-PGD protein and A549 cell lysate protein. The results of the in vitro assays supported the in silico findings. In order to gain insight into the anticancer activity of the aforementioned compounds, they were subjected to CLC-Pred, an in silico cytotoxicity browsing tool, which proved their anticancer activity against several cancer cell lines at Pa > 0.5. Additionally, a confirmation for in silico cytotoxicity was made by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for commercially available hits syringaresinol and cleomiscosin A against lung cancer (A549) cells. The results demonstrated that syringaresinol has an IC50 value of 36.9 µg/mL, while cleomiscosin A has an IC50 value of 133 µg/mL. After MTT, flow cytometry analysis confirmed that compounds induced apoptosis in A549 cells in a dose-dependent manner. This study suggested that the respective lignan compounds can serve as lead candidates for lung cancer therapy via 6-PGD inhibition. Furthermore, in vivo experiments need to be conducted to confirm their efficacy.

Clinical profile and screening of exon 6 and 14 of ABCB4 gene in obstetric cholestasis patients at a tertiary care hospital in Rawalpindi, Pakistan.

OBJECTIVE: Intrahepatic Cholestasis of Pregnancy (ICP) is a rare pregnancy specific disorder. Genetic variants of ABCB4 gene increase ICP risk. This study was conducted to determine frequency of ICP cases presented at a tertiary care hospital in Rawalpindi, Pakistan and to screen for genetic variants of exon 6 and 14 of ABCB4 gene in ICP cases. METHODS: This analytical study included ICP patients presenting at Department of Gynaecology and Obstetrics, Holy Family Hospital Rawalpindi, from February 2017 to May 2017. Sanger's sequencing was performed using genomic DNA extracted from blood samples of patients and controls. RESULTS: Twenty pregnant women out of 1150 (1.74%) had ICP and were enrolled during study period. Overall (19/20) 95% patients had pruritus and among them (8/20) 40%, (4/20) 20% and (2/20) 10% had a history of miscarriages, stillbirths and familial ICP respectively. Genetic analysis revealed an already reported variant i.e., c.504C>T in exon 6 in thirteen patients and a novel variant i.e., c.1686A>G in exon 14 in five patients. Both variants were not present in controls. In silico analysis suggested that both variants might affect pre-mRNA splicing of ABCB4 transcript. CONCLUSIONS: ICP had a frequency of 1.74% among pregnant women. Identification of a novel heterozygous variant in five patients and an already reported variant in thirteen patients reaffirms genetic heterogeneity and role of ABCB4 in ICP etiology.

Social support and health related quality of life among pregnant women.

OBJECTIVE: To explore the relationship between social support and health-related quality of life among pregnant women. METHODS: This cross-sectional study was conducted at City Hospital, Kashrote, and District Headquarters Hospital in the city of Gilgit, Pakistan, from January to March2017.Pregnant women presenting to the two hospitals were approached for data collection. The chronological age range of the sample was 15-35 years and age of pregnancy in months were included. Social support of pregnant women was measured through multidimensional scale of perceived social support and health-related quality of life was measured through medical outcomes study questionnaire.. RESULTS: Of the 120 participants, 66(55%) were from the City Hospital, Kashrote, and 54(45%) from the District Headquarters Hospital. Subjects who were in their first trimester of pregnancy were 72(60%), while 25(20.8%) were in their second trimester and 23 (19.2%) in the third. Of the total, 80(66.66%) were educated women and 40 (33.34%) were illiterate. Subjects with a history of physical illness were 76 (63.30%) while 60 (50%) suffered from psychological illness too. There was a significantly positive relationship between social support and health-related quality of life among the pregnant subjects (p<0.05). CONCLUSIONS: More social support, can increase the health-related quality of life of pregnant women.

Comparative Study of Carcinoembryonic Antigen Tumor Marker in Stomach and Colon Cancer Patients in Khyber Pakhtunkhwa.

BACKGROUND: Due to the increase in morbidity and mortality rate, cancer has become an alarming threat to the human population worldwide. Since cancer is a progressive disorder, timely diagnosis would be helpful to prevent/stop cancer from progressing to severe stage. In Khyber Pakhtunkhwa, Pakistan, most of the time, tumors are diagnosed with endoscopy and biopsy; therefore rare studies exist regarding the diagnosis of gastrointestinal (GIT) carcinomas based on tumor markers, especially CEA. OBJECTIVES: This study made a comparative analysis of CEA in admitted hospitalized stomach and colon cancer patients diagnosed as GIT with biopsy. MATERIALS AND METHODS: In this study, a total of 66 cases were included. The level of CEA was determined in the blood of these patients using ELISA technique. RESULTS: Out of 66 patients, the level of CEA was high in 59.1% of the total, 60.7% in colon cancer patients and 57.9 % in stomach cancer patients. Moreover, the incidence of colorectal and stomach cancer was greater in males as compared to females. Patients were more of the age group of 40- 60 and the level of CEA was comparatively higher in patients (51.5%) with histology which was moderately differentiated, than patients with well differentiated and poorly differentiated tumor histology. CONCLUSIONS: CEA level was high in more than 50% of the total patients. Moreover, CEA exhibited higher sensitivity for colon than stomach cancer.

Level and evaluation of tumor marker CA-125 in ovarian cancer patients in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Due to the increase in morbidity and mortality rate, cancer has become an alarming threat to the human population worldwide. Since cancer is a progressive disorder, timely diagnosis is necessary to prevent/stop cancer from progressing to a severe stage. In Khyber Paktunkhwa, Pakistan, many tumors are diagnosed with endoscopy and biopsy; rare studies exist regarding the diagnosis and evaluation of ovarian cancer, based on tumor markers like CA-125. OBJECTIVES: The objectives of this study were to investigate and evaluate levels of CA-125 in hospitalized ovarian cancer patients. MATERIALS AND METHODS: In this study, a total of 63 admitted patients having ovarian cancer by biopsy were included. The level of CA-125 was determined in the blood of these patients using ELISA technique. RESULTS: Out of 63 patients, the level of CA-125 was high in 52% . The affected individuals were more in the group of 40-60 and the level of CA-125 was comparatively higher in patients having moderately differentiated histology than those having well differentiated and poorly differentiated tumor histology. Moreover, the highest level of CA-125 was present among the patients having serous subtype of carcinoma and the common stage of carcinoma was stage II followed by stage III, I and IV. CONCLUSIONS: CA-125 level was high in more than 50% of the total patients. Moreover, CA-125 elevation was more common in serous subtype and stage II cancer patients.