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The present study was designed to improve the aqueous solubility and dissolution rate of citreorosein, a poorly water soluble drug, by producing its nanoparticles (NPs) using an “antisolvent precipitation with a syringe pump” (APSP) method. Various process parameters including flow rate, stirring speed, temperature, drug concentration and solvent-antisolvent ratio were investigated and optimized to obtain the smallest particle size of citreorosein. The prepared NPs were subjected to different analytical techniques such as SEM, FTIR, XRD and DLS. The NPs were tested for different dissolution parameters including difference (f1) and similarity factors (f2), dissolution efficiency (DE) and mean dissolution time (MDT). The antioxidant potential of citreorosein and its NPs were evaluated using DPPH, ABTS, and FRAP models, while their cytotoxicity was tested on various cancer cell lines such as breast (MDA-MB-231), lung (A549) and liver (HepG2), using the MTT assay. The specificity of the test compound was investigated against two normal human primary epithelial cells including renal (HRPTEpiC) and alveolar (HPAEpiC) cells. DLS analysis revealed that the prepared NPs were less than 200 nm in size while SEM and XRD confirmed their rod shape and amorphous nature, respectively. Citreorosein-NPs exhibited enhanced solubility and dissolution rate in all medias as compared to pure citreorosein. The NPs displayed significant antioxidant effects against ABTS, DPPH and FRAP models, with IC50 values of22.6, 21.45 and 27.30 µg/ml, respectively. Moreover, it showed significant cytotoxic activity against MDA-MB-231, A549 and HepG2 cells, with IC50 values of 3.45, 4.5 and 6.23 µg/ml, respectively. Furthermore, the NPs demonstrated high selectivity index values for the aforementioned cell lines as compared to reference drugs. This study demonstrated that APSP method successfully produced citreorosein-NPs and hence, showed better aqueous solubility, dissolution rate, antioxidant and cytotoxic activities than the pure compound.

Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer.Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1 mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2 v/v) to 0.5 mL of plasma. The internal standard solution (artemisinin 500 ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 μL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 μL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV.Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines.Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer’s plasma.

Genome-wide association studies have greatly increased the number of T2DM associated risk variants but most of them have focused on populations of European origin. There is scarcity of such studies in developing countries including Pakistan. High prevalence of T2DM in Pakistani population prompted us to design this study. We have devised a two stage (the discovery stage and validation stage) case-control study in Pashtun ethnic population in which 500 T2DM cases and controls each have been recruited to investigate T2DM genetic risk variants. In discovery stage Whole Exome Sequencing (WES) was used to identify and suggest T2DM pathogenic SNPs, based on SIFT and Polyphen scores; whereas in validation stage the selected variants were confirmed for T2DM association using MassARRAY genotyping and appropriate statistical tests. Results of the study showed the target positive association of rs1801282/PPARG (OR = 1.24, 95%Cl = 1.20-1.46, P = 0.010), rs745975/HNF4A (OR = 1.30, 95%Cl = 1.06-1.38, P = 0.004), rs806052/GLIS3 (OR = 1.32, 95%Cl = 1.07-1.66, P = 0.016), rs8192552/MTNR1B (OR = 1.53, 95%Cl = 0.56-1.95, P = 0.012) and rs1805097/IRS-2 (OR = 1.27, 95%Cl = 1.36-1.92, P = 0.045), with T2DM; whereas rs6415788/GLIS3, rs61788900/NOTCH2, rs61788901/NOTCH2 and rs11810554/NOTCH2 (P>0.05) showed no significant association. Identification of genetic risk factors/variants can be used in defining high risk subjects assessment, and disease prevention.

Genome-wide association studies have greatly increased the number of T2DM associated risk variants but most of them have focused on populations of European origin. There is scarcity of such studies in developing countries including Pakistan. High prevalence of T2DM in Pakistani population prompted us to design this study. We have devised a two stage (the discovery stage and validation stage) case-control study in Pashtun ethnic population in which 500 T2DM cases and controls each have been recruited to investigate T2DM genetic risk variants. In discovery stage Whole Exome Sequencing (WES) was used to identify and suggest T2DM pathogenic SNPs, based on SIFT and Polyphen scores; whereas in validation stage the selected variants were confirmed for T2DM association using MassARRAY genotyping and appropriate statistical tests. Results of the study showed the target positive association of rs1801282/PPARG (OR = 1.24, 95%Cl = 1.20–1.46, P = 0.010), rs745975/HNF4A (OR = 1.30, 95%Cl = 1.06–1.38, P = 0.004), rs806052/GLIS3 (OR = 1.32, 95%Cl = 1.07–1.66, P = 0.016), rs8192552/MTNR1B (OR = 1.53, 95%Cl = 0.56–1.95, P = 0.012) and rs1805097/IRS-2 (OR = 1.27, 95%Cl = 1.36–1.92, P = 0.045), with T2DM; whereas rs6415788/GLIS3, rs61788900/NOTCH2, rs61788901/NOTCH2 and rs11810554/NOTCH2 (P>0.05) showed no significant association. Identification of genetic risk factors/variants can be used in defining high risk subjects assessment, and disease prevention.

Free radicals, mostly consist of reactive oxygen species, are generated in human body by several exogenous and endogenous systems. Overproduction of free radicals is known to cause several degenerative disorders including cancer. The aim of this study is to synthesize silver nanoparticles (AgNPs) using root extract of Reynoutria japonica and to investigate its antioxidant and cytotoxic potential. AgNPs were synthesized by green approach and subsequently characterized using UV–vis spectroscopy, SEM, TEM, FTIR, XRD, EDS and DLS. The antioxidant activity was investigated using DPPH, FRAP, H2O2, and ABTṠ+ radical scavenging assays while the cytotoxic effect was assessed using different human cancer cell lines including lung (A549), liver (Hep-G2) and breast (MDA-MB-231) by MTS assay. Moreover, the specificity of NPs was assessed against two normal human cell lines e.g. alveolar and renal primary epithelial cells (HPAEpiC and HRPTEpiC). The UV–vis spectra confirmed the synthesis of AgNPs by producing a characteristic peak at 410 nm. Further analysis confirmed that AgNPs were crystalline in nature, predominantly spherical in shape, with an average width and area of 17.34 nm and 164.46 nm2, respectively. DLS analysis revealed that NPs possess a high negative zeta potential value (−28.5 mV), thus facilitating its electrostatic stabilization. AgNPs showed dose dependent antioxidant activity against DPPH, FRAP, H2O2 and ABTS with IC50 values 19.25, 22.45, 24.20 and 18.88 µg/ml, respectively. The AgNPs depicted significant cytotoxic effects against A549, Hep-G2 and MDA-MB-231 cell lines with IC50 values of 4.5, 5.1 and 3.46 µg/ml, respectively. Moreover, the NPs exhibited highest selectivity index (>2.0) for A549, Hep-G2 and MDA-MB-231, confirming its specificity towards cancer cell lines. In conclusion, AgNPs prepared from root extract of R. japonica possess strong antioxidant and cytotoxic potential which suggests that they should be investigated further in order to develop safe and effective antioxidant and/or cytotoxic formulations.

Background and ObjectiveThe standards of living, improvement in public health, and medical care in Pakistan are increasing day by day, health-related quality of life (HRQoL) has been increasingly acknowledged in various patient's reported outcomes in Pakistan. However, a large-scale general population-based study on assessing HQRoL in Pakistan was not conducted. Therefore, this study aimed to evaluate HRQoL for the general Pakistani population.Material and MethodsA cross-sectional study with a population sample (n = 16,672) was selected from all Pakistan provinces using a stratified sampling approach. The EQ-5D-3L tool was used to measure the HRQoL of the general population of Pakistan. The descriptive and inferential statistics have been done by using SPSS version 20.ResultsOverall, 121 health states were reported in this study. EQ-5D index and EQ-VAS scores were 0.74 ± 0.32 and 0.75 ± 0.25, respectively. The percentage of people responding to any problems increased with age. Males have better health as compared to females in all age groups. All demographics were significantly associated (P < 0.01) with the mean EQ5D index and VAS scores except residence (p > 0.05). The regression model reported that age was the best predictor of the EQ-5D index scores after adjusting for the covariates (beta = 0.19; p < 0.001). This study provides Pakistani population HRQoL data measured by the EQ-5D tool, based on a national representative sample.ConclusionThe current study concluded that Age, City, Gender, Education, Occupation, Residence, and House occupancy are significantly affecting HRQOL. The socioeconomically deprived groups and females have inferior health status than more advantaged. The trends detected in high-income nations were usually similar to Pakistan.

Objective. Recent GWAS largely conducted in European populations have successfully identified multiple genetic risk variants associated with Type 2 Diabetes Mellitus (T2DM). However, the effects conferred by these variants in the Pakistani population have not yet been fully elucidated. The objective of this study was to examine European GWAS- identified T2DM risk variants in the Pakistani Pashtun population to better understand the shared genetic basis of T2DM in the European and Pakistani cohorts. Methodology. A total of 100 T2DM patients and 100 healthy volunteers of Pashtun ethnicity were enrolled in this study. Both groups were genotyped for 8 selected single nucleotide polymorphisms (SNPs) using the Sequenom MassARRAY® platform. The association between selected SNPs and T2DM was determined by using appropriate statistical tests. Results. Of the 8 studied SNPs, 5 SNPs, SLC30A8/ rs13266634 (p=0.031, OR=2.13), IGF2BP2/ rs4402960 (p=0.001, OR=3.01), KCNJ11/ rs5219 (p=0.042, OR=1.78), PPARG/ rs1801282 (p=0.042, OR=2.81) and TCF7L2/ rs7903146 (p=0.00006, 3.41) had a significant association with T2DM. SNP GLIS3/ rs7041847 (p=0.051, OR=2.01) showed no sufficient evidence of association. SNPs KCNQ1/ rs2237892 (p=0.140, OR=1.61) and HHEX/IDE/ s1111875 (p=0.112, OR=1.31) showed opposite allelic effects and were not validated for T2DM risk in the study population. Among the studied SNPs, TCF7L2/ rs7903146 showed the most significant association. Conclusion. Our study finding indicates that selected genome-wide significant T2DM risk variants previously identified in European descent also increase the risk of developing T2DM in the Pakistani Pashtun population.

Traditionally, Natural Organic Matter (NOM) derived from cretaceous rocks has been used for treatment of various ailments such as diabetes, inflammation and skin infections. This study evaluated the antimicrobial, antioxidant and anti-inflammatory activities of natural organic matter obtained from cretaceous shales. The shales were collected from Lumshiwal formation; located north of the main Kala Chitta range in district Nowshera-Pakistan. Isolation was done by sonicating crushed rock sample with chloroform, methanol and acetone (70: 15: 15 v/v, respectively). Antibacterial and antifungal activity of sample was determined by agar well diffusion and Agar slanting methods, respectively. In vitro anti-inflammatory activity was performed using cyclooxygenase-2 and 5-lipoxygenase enzymes. Antioxidant activity was assessed for scavenging of DPPH, superoxide anions, hydroxyl radicals, and hydrogen peroxide. In vivo anti-inflammatory activity was performed using “Carrageenan-induced paw edema model”. The sample showed significant antibacterial activity against Salmonella typhi, Pseudomonas aeruginosa and Escherichia coli with MIC values 0.82, 0.87 and 0.79 mg/ml, respectively. Considerable inhibition was observed against Bacillus subtilis (MIC; 0.93 mg/ml) and Staphylococcus aureus (MIC; 1.12 mg/ml) when compared with Imipenem as a standard. Moreover, the sample displayed significant antifungal activity against Alternaria alternata and Fusarium solani with MIC values of 0.60 and 0.68 mg/ml, respectively. Both COX-2 (IC50 31.34 µg/ml) and 5-LOX (IC50 38.45 µg/ml) enzymes were inhibited by NOM in a concentration-dependent manner. In addition, the NOM exhibited significant free radical scavenging, especially against DPPH and superoxide anions; and a moderate effect on hydroxyl and hydrogen peroxide scavenging. In vivo anti-inflammatory activity revealed that the edema volume was significantly (P

Cannabis abuse is a common public health issue and may lead to considerable adverse effects. Along with other effects, the dependence on cannabis consumption is a serious problem which has significant consequences on biochemical and clinical symptoms. This study intends to evaluate the harmful effects of the use of cannabis on thyroid hormonal levels, cardiovascular indicators, and psychotic symptoms in the included patients. This prospective multicenter study was conducted on cannabis-dependent patients with psychotic symptoms (n = 40) vs. healthy control subjects (n = 40). All participants were evaluated for psychiatric, biochemical, and cardiovascular physiological effects. Patients were selected through Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria and urine samples, exclusively for the evaluation of cannabis presence. Serum thyroid stimulating hormone (TSH), T3, and T4 levels were measured using the immunoassay technique. Patients were assessed for severity of depressive, schizophrenic, and manic symptoms using international ranking scales. Various quantifiable factors were also measured for the development of tolerance by cannabis. Among the patients of cannabis abuse, 47.5% were found with schizophrenia, 20% with schizoaffective symptoms, 10% with manic symptoms, and 22.5% with both manic and psychotic symptoms. In the group–group and within-group statistical analysis, the results of thyroid hormones and cardiovascular parameters were non-significant. The psychiatric assessment has shown highly significant (p < 0.001) difference of positive, negative, general psychopathology, and total scores [through Positive and Negative Syndrome Scale (PANSS) rating scales] in patients vs. the healthy control subjects. The study revealed that cannabis abuse did not significantly alter thyroid hormones and cardiovascular parameters due to the development of tolerance. However, the cannabis abuse might have a significant contributing role in the positive, negative, and manic symptoms in different psychiatric disorders.

Resistance to antimicrobial agents are rendering therapies ineffective around the globe, leading to increased mortality and treatment cost. Likewise, non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics possess several side effects particularly peptic ulcer and gastrointestinal problems. Metallic nanoparticles significantly enhances the therapeutic efficacy of natural extracts owing to improved bioavailability thereby lowering the dose and side effects. The purpose of this research was to investigate the efficacy of gold nanoparticles (AuNPs). In this study, Sambucus wightiana whole plant aqueous extract was used for rapid and eco-friendly synthesis of AuNPs. They were characterized by various analytical techniques including UV–Visible spectroscopy (UV–Vis), transmission and scanning electron microscopy (TEM and SEM), energy dispersive X-ray spectroscopy (EDX), Zetasizer, X-ray diffractometer (XRD) and Fourier transform infra-red spectroscopy (FTIR). The UV–Vis spectra revealed a distinct absorption peak at 539 nm; TEM and SEM images confirmed the formation of heterogeneously dispersed AuNPs with an average area of 152.77 nm2 and width of 15.96 nm. The AuNPs showed significant inhibitory zones against Escherichia coli (25 mm), Staphylococcus epidermis (23 mm) and Salmonella enteritidis (18 mm) with MIC values 0.13, 0.11 and 0.16 mg/ml, respectively. Among fungal strains it showed highest percent inhibition against Fusarium solani (90%) and Microsporum canis (80%) with MIC values 0.02 and 0.01 mg/ml, respectively. It showed maximum anti-inflammatory activity (43.70, 48.80 and 57.08%) at 20 mg/kg dose at both early and late hours of inflammation. Likewise, in vitro models depicted concentration dependent inhibition of 5-LOX and COX-2 enzymes. AuNPs showed highest antioxidant activity (68.7% inhibition) at 1000 µg/ml, compared to ascorbic acid that showed 77.8% inhibition at the same concentration. Similarly, it exhibited significant (P ≤ 0.001) dose dependent analgesic effect with maximum inhibition (56.22%) at 20 mg/kg. In conclusion, the above findings suggest that AuNPs should be studied further in order to develop safe and effective formulations.

Background and objectives: COVID-19 patients exhibit a broad range of manifestations, presenting with a flu-like respiratory tract infection that can advance to a systemic and severe disease characterized by pneumonia, pulmonary edema, severe damage to the airways, and acute respiratory distress syndrome (ARDS, causing fatality in 70% of COVID-19 cases). A ‘cytokine storm’ profile is found in most severely influenced COVID-19 patients. The treatment protocol of the disease also includes tocilizumab, which is a humanized monoclonal antibody used to treat autoimmune and inflammatory conditions. This study was designed (1) to assess the role of tocilizumab in COVID-19 patients regarding therapeutic efficacy through evaluation of cytokine release syndrome (CRS) resolution and anticoagulant effect, analyzing clinical safety via monitoring of associated adverse effects profile; and (2) to compare the clinical safety and therapeutic efficacy of institutional treatment regimen (alone) versus tocilizumab added to an institutional treatment module in COVID-19 patients. Materials and Methods: In this study, the endpoints parametric assessment of severely diseased patients of COVID-19 was performed (total n = 172, control group (institutional protocol treatment provided), n = 101 and test group (tocilizumab provided), n = 71) at the Khyber Teaching Institution, MTI, Peshawar. The assessments were compared using non-parametric analyses at baseline and after a follow-up of 12–18 days until the patient discharged or expired. Results: Results of the study revealed an insignificant difference among the control vs. test group in resolving inflammatory parameters (C-reactive protein (CRP) 21.30 vs. 50.07; p = 0.470, ferritin 482.9 vs. 211.5; p = 0.612, lactate dehydrogenase (LDH) 29.12 vs.18.8; p = 0.0863, and D-dimer 464 vs.164.4; p = 0.131). However, a statistically significant difference was found between the control group and test group regarding coagulation parameters (international normalized ratio (INR) 0.12 vs. −0.07; p ≤ 0.001; activated partial thromboplastin time (aPTT) 0.42 vs. −1.16; p ≤ 0.001; prothrombin time (PT) 0.31 vs. −0.96; p ≤ 0.001; platelet count −12.34 vs. −1.47; p = 0.012) and clinical survival rate (89.10 vs. 90.14; p < 0.001). Furthermore, there was significantly higher infection rates and raised alanine aminotransferase (ALT) and alkaline phosphatase (ALP) associated with the tocilizumab group as compared to those receiving institutional treatment (bacterial infections: 0.99% vs. 15.49%; p ≤ 0.01, ALT: 3.96% vs. 28.16%; p ≤ 0.01, ALP: 1.98% vs. 22.53%; p ≤ 0.01). Conclusions: From this study, it was concluded that tocilizumab can be a better drug of choice in terms of efficacy, particularly in resolving coagulopathy in severe COVID-19 patients.

Leishmaniasis is affirmed as a category one disease (most emerging and unmanageable) by the World Health Organization (WHO), affecting 98 countries with an annual global incidence of ~1.2 million cases. Options for chemotherapeutic treatment are limited due to drug resistance and cytotoxicity. Thus, the search for new chemical compounds is instantly desirable. In this study, we used two compounds, i.e., 10-hydroxy chondrofoline and tafenoquine, for their antileishmanial activity against L. tropica (HTD7). First, the cytotoxicity assay of the test compounds against THP-1 cells was carried out, and these compounds were found safe. Intra-THP-1 amastigote activity (in vitro) was performed, which was then followed by the in vivo activity of 10-hydroxy chondrofoline in the murine cutaneous leishmaniasis (CL) model. A total of three concentrations were used, i.e., 25, 50, and 100 µM, to check the in vitro activity of the test compounds against the amastigotes. 10-hydroxy chondrofoline was found to be the most potent compound in vitro (and thus was selected for in vivo studies) with an LD50 value of 43.80 µM after 48 h incubation, whilst tafenoquine had an LD50 value of 53.57 µM. In vivo activity was conducted by injecting 10-hydroxy chondrofoline in the left hind foot of the infected BALB/c mice, where it caused a statistically significant 58.3% (F = 14.18; p = 0.002) reduction in lesion size (0.70 ± 0.03 mm) when compared with negative control (1.2 ± 0.3 mm).

Treatment of microbial infections and inflammatory conditions have many challenges in terms of efficacy and safety issues. Novel approaches such as nanoparticles based drug delivery system have shown promising results to solve some of these problems. The aim of this study was to exploit the efficacy of the synthesized silver nanoparticles. In this study, silver nanoparticles (AgNPs) were biosynthesized using root extract (aqueous) of Duchesnea indica. They were characterized using different techniques such as, ultraviolet–visible (UV–Vis) spectrophotometry, transmission and scanning electron microscopy (TEM and SEM), X-ray diffractometer (XRD), energy dispersive X-ray spectroscopy (EDX), fourier-transform infrared spectroscopy (FTIR) and zetasizer. The UV–Vis spectra gave a characteristic peak at 423 nm; XRD confirmed its crystalline structure; FTIR confirmed the involvement of phytochemicals in their capping and reduction; TEM images confirmed their spherical shape with average width of 20.49 nm and average area of 319.25 nm2. Various biological activities were performed on these NPs, such as antimicrobial, anti-inflammatory, analgesic and muscle relaxant, which showed significant results as follow. Among bacterial strains, Salmonella typhi (MIC: 0.01 mg/ml) and Escherichia coli (MIC: 0.01 mg/ml), while among that of fungal Microsporum canis (MIC: 0.53 mg/ml) and Alternaria alternata (MIC: 0.51 mg/ml) were most susceptible. The AgNPs showed maximum anti-inflammatory activity (46.15 and 56.85%) at 20 mg/kg after 3 and 5 h of drug administration, comparable to that of standard. In-vivo model exhibited concentration dependent inhibition of both COX-2 and 5-LOX enzymes. Similarly, it exhibited maximum analgesic activity (54.24%) at 20 mg/kg dose after 60 min. of pain induction. Furthermore, they depicted maximum muscle relaxation (P

Mirtazapine is a tetracyclic anti-depressant with poor water solubility. The aim of this study was to improve the dissolution rate of mirtazapine by delivering the drug as a liquisolid compact. Central composite design (CCD) was employed for the preparation of mirtazapine liquisolid compacts. In this, the impacts of two independent factors, i.e., excipient ratio (carrier:coating) and different drug concentration on the response of liquisolid system were optimized. Liquisolid compacts were prepared using propylene glycol as a solvent, microcrystalline cellulose as a carrier, and silicon dioxide (Aerosil) as the coating material. The crystallinity of the formulated drug and the interactions between the excipients were examined using X-ray powder diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR), respectively. The dissolution study for the liquisolid compact was carried out as per FDA guidelines. The results showed loss of crystallinity of the mirtazapine in the formulation and was completely solubilized in non-volatile solvent and equally dispersed throughout the powder system. Moreover, drug dissolution was found to be higher in liquisolid compacts than the direct compressed conventional tablets (of mirtazapine). The liquisolid technique appears to be a promising approach for improving the dissolution of poorly soluble drugs like mirtazapine.

Human leukocyte antigen (HLA) system is the most polymorphic and gene dense region of human DNA that has shown many disease associations. It has been further divided into HLA classes I, II, and III. Polymorphism in HLA class II genes has been reported to play an important role in the pathogenesis of type 1 diabetes (T1D). It also showed association with T2D in different ethnic populations. However, a little is known about the relationship of HLA class I gene polymorphism and T2D. This study has evaluated the association of HLA-B (class I gene) variants with T2D in Pashtun ethnic population of Khyber Pakhtunkhwa. In the first phase of the study, whole exome sequencing (WES) of 2 pooled DNA samples was carried out, and DNA pools used were constructed from 100 diabetic cases and 100 control subjects. WES results identified a total of n=17 SNPs in HLA-B gene. In the next phase, first 5 out of n=17 reported SNPs were genotyped using MassARRAY® system in order to validate WES results and to confirm association of selected SNPs with T2D. Minor allele frequencies (MAFs) and selected SNPs×T2D association were determined using chi-square test and logistic regression analysis. The frequency of minor C allele was significantly higher in the T2D group as compared to control group (45.0% vs. 13.0%) (p=0.006) for rs2308655 in HLA-B gene. No significant difference in MAF distribution between cases and controls was observed for rs1051488, rs1131500, rs1050341, and rs1131285 (p>0.05). Binary logistic regression analyses showed significant results for SNP rs2308655 (OR=2.233, CI 95%=1.223‐4.077, and p=0.009), while no considerable association was observed for the other 4 SNPs. However, when adjusted for these variants, the association of rs2308655 further strengthened significantly (adjusted OR=7.485, CI 95%=2.353‐23.812, and p=0.001), except for rs1131500, which has no additive effect. In conclusion, the finding of this study suggests rs2308655 variant in HLA-B gene as risk variant for T2D susceptibility in Pashtun population.

Suicide, a deliberate act of self-harm with the intention to die, is an emerging health concern but, unfortunately, the most under-researched subject in Pakistan, especially in Khyber Pukhtunkhwa (KPK). In this study, we aimed to identify risk factors that can be associated with suicidal behavior (SB) and to evaluate the prevailing treatment practices for therapeutic efficacy and drug-related problems (DRPs) in psychotic patients among the local population of KPK. A prospective, multicenter study was conducted for suicidal cases admitted to the study centers by randomized sampling. Socio-demographics and data on suicidal behavior were assessed using the Columbia-Suicide Severity Rating Scale (C-SSRS), socioeconomic condition by Kuppuswamy socioeconomic scale (KSES) and treatment adherence by Morisky Medication-Taking Adherence Scale (MMAS-4). Drug-related problems and the therapeutic efficacy of prevailing treatment practices were assessed at baseline and follow-up after 3 months of treatment provided. Regarding suicidality (N = 128), females reported more ideations (63.1%), while males witnessed more suicidal behavior (66.6%, p < 0.001). Suicide attempters were mostly married (55.6%, p < 0.002); highly educated (53.9%, p = 0.004); dissatisfied with their life and had a previous history (p < 0.5) of suicide attempt (SA) (20.6%), self-injurious behavior (SIB) (39.7%) and interrupted (IA) or aborted attempts (AA) (22.2%). A greater improvement was observed in patients receiving combination therapy (p = 0.001) than pharmacotherapy (p = 0.006) or psychotherapy (p = 0.183), alone. DRPs were also detected, including drug-selection problems (17.88%), dose-related problems (20.64%), potential drug–drug interactions (24.31%), adverse drug reactions (11.46%) and other problems like inadequate education and counseling (21.55%). Furthermore, it was also found that psychotic patients with suicidal ideations (SI) were significantly (p = 0.01) more adherent to the treatment as compared to those with suicidal attempts. We concluded that suicide attempters differed significantly from patients with suicidal ideations in psychotic patients and presented with peculiar characteristics regarding socio-demographic factors. A combination of therapies and adherence to the treatment provided better outcomes, and targeted interventions are warranted to address drug-related problems.

Medicinal plants are known for their diverse use in the traditional medicine of the Himalayan region of Pakistan. The present study is designed to investigate the anticancer and antimicrobial activities of Prunus cornuta and Quercus semicarpifolia. The anticancer activity was performed using cancerous human cell lines (HepG2, Caco-2, A549, MDA-MB-231, and NCI-H1437 carcinoma cells), while the antimicrobial activity was conducted with the agar-well diffusion method. Furthermore, toxicity studies were performed on alveolar and renal primary epithelial cells. Initially, different extracts were prepared by maceration techniques using n-hexane, chloroform, ethyl acetate, butanol, and methanol. The preliminary phytochemical screening showed the presence of secondary metabolites such as alkaloids, tannins, saponins, flavonoids, glycosides, and quinones. The chloroform extract of P. cornuta (PCC) exhibited significant inhibitory activity against Acinetobacter baumannii (16 mm) and Salmonella enterica (14.5 mm). The A. baumannii and S. enterica strains appeared highly susceptible to n-hexane extract of P. cornuta (PCN) with an antibacterial effect of 15 mm and 15.5 mm, respectively. The results also showed that the methanolic extracts of Quercus semecarpifolia (QSM) exhibited considerable antibacterial inhibitory activity in A. baumannii (18 mm), Escherichia coli (15 mm). The QSN and QSE extracts also showed good inhibition in A. baumannii with a 16 mm zone of inhibition. The Rhizopus oryzae strain has shown remarkable mycelial inhibition by PCM and QSN with 16 mm and 21 mm inhibition, respectively. Furthermore, the extracts of P. cornuta and Q. semicarpifolia exhibited prominent growth inhibition of breast (MDA-MB-231) and lung (A549) carcinoma cells with 19–30% and 22–39% cell viabilities, respectively. The gut cell line survival was also significantly inhibited by Q. semicarpifolia (24–34%). The findings of this study provide valuable information for the future development of new antibacterial and anticancer medicinal agents from P. cornuta and Q. semicarpifolia extracts.

The Aquifoliaceae is an important family and has been used traditionally for some time. One of the members of this family is the Ilex dipyrena wall, which itself possesses a potential medicinal importance. This plant is traditionally used for the treatment of various ailments including pain, swelling, burns, and fever. The current study was designed to screen out the antioxidant and analgesic potential of this plant and to verify its traditional uses, along with its phytochemical profile. Extracts were subjected to antioxidant, analgesic, and phytochemical analysis using DPPH, chemical-induced (acetic acid and formalin) nociception models and GC-MS analysis, respectively. The leaf, bark, and fruit extracts showed significant antioxidant activity compared to that of standard. Likewise, all the extracts demonstrated significant (p < 0.01) analgesic activity in a mice model. In acetic acid induced analgesia, the leaf, bark, and fruit extracts caused 51.64, 56.13 and 59.52% inhibition, respectively at a dose of 100 mg/kg while at 200 mg/kg it showed 83.01, 71.69 and 75.47% inhibition, respectively. In Formalin-induced paw-licking assay, fruit extract showed 59.42 and 64.19% inhibition at 200 mg/kg dose in the first and second phase, respectively. The GC-MS analysis revealed the presence of cathinone, phenylpropanolamine, dl-phenylephrine, amphetamine, myristic acid, and palmitic acid. Results of the study suggest that crude extracts from different parts of this plant may be a useful source for the development of novel analgesics. However, further investigation in terms of isolation of bioactive compounds and their toxicological evaluations are needed to validate the observed results.