Microwave-Assisted Physically Cross-Linked Chitosan-Sodium Alginate Hydrogel Membrane Doped with Curcumin as a Novel Wound Healing Platform.

This project purposes to develop chitosan and sodium alginate-based hydrogel membranes loaded with curcumin through microwave-based physical cross-linking technique and its evaluation for wound healing potential. For the purpose, curcumin-loaded chitosan and sodium alginate membranes were developed using microwave at fixed frequency of 2450 MHz, power 350 W for 60 s, and tested for their physicochemical attributes like swelling, erosion, surface morphology, drug content, and in vitro drug release. The membranes were also subjected to tensile strength and vibrational and thermal analysis followed by testing in vivo on animals. The results indicated that microwave treatment significantly enhanced the swelling ability, reduced the erosion, and ensured smooth surface texture with optimal drug content. The drug was released in a slow fashion releasing total of 41 ± 4.2% within 24-h period with a higher tensile strength of 16.4 ± 5.3 Mpa. The vibrational analysis results revealed significant fluidization of hydrophilic domains and defluidization of hydrophobic domains which translated into a significant rise in the melting temperature and corresponding enthalpy which were found to be 285.2 ± 3.2 °C and 4.89 ± 1.4 J/g. The in vivo testing revealed higher percent re-epithelialization (75 ± 2.3%) within 14 days of the treatment application in comparison to only gauze and other treatments applied, with higher extent of collagen deposition having well-defined epidermis and stratum corneum formation. The microwave-treated chitosan-sodium alginate hydrogel membranes loaded with curcumin may prove to be another alternative to treat skin injuries. Graphical Abstract.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

Ophthalmo-genetic analysis of Pakistani patients with nonsyndromic oculocutaneous albinism through whole exome sequencing.

Oculocutaneous albinism (OCA) is a disorder of defective melanin biosynthesis that is characterized by hypo-pigmentation of skin, hair and retinal pigment epithelium. Phenotypically, OCA patients exhibit white milky skin, whitish to golden hair and deterioration of retinal cells. Until recently, genetic studies have reported seven causative genes (TYR, TYRP1, OCA2, SLC45A2, SLC24A2, C10ORF11 and MCIR) and an uncharacterized OCA5 locus. Herein we present the medico-genetic study of three Pakistani patients inheriting autosomal recessive OCA. Whole exome sequencing, followed by Sanger DNA sequencing for segregation analysis, revealed recurrent mutations c.346C>T (p.Arg116*) and c.1255G>A (p.Gly419Arg) (family A and B respectively) in TYR gene, while the patient from family C did not reveal any known gene mutation, which suggests the involvement of some novel genetic factor. It is the first report of mapping c.346C>T mutation in a Pakistani patient. Our study further extends the evidence of genetic hotspots regions in TYR gene causing OCA in Pakistani population.

The Molecular Genetics of Autosomal Recessive Nonsyndromic Intellectual Disability: a Mutational Continuum and Future Recommendations.

Intellectual disability (ID) is a clinical manifestation of the central nervous system without any major dysmorphologies of the brain. Biologically it affects learning capabilities, memory, and cognitive functioning. The basic defining features of ID are characterized by IQ<70, age of onset before 18 years, and impairment of at least two of the adaptive skills. Clinically it is classified in a syndromic (with additional abnormalities) and a nonsyndromic form (with only cognitive impairment). The study of nonsyndromic intellectual disability (NSID) can best explain the pathophysiology of cognition, intelligence and memory. Genetic analysis in autosomal recessive nonsyndrmic ID (ARNSID) has mapped 51 disease loci, 34 of which have revealed their defective genes. These genes play diverse physiological roles in various molecular processes, including methylation, proteolysis, glycosylation, signal transduction, transcription regulation, lipid metabolism, ion homeostasis, tRNA modification, ubiquitination and neuromorphogenesis. High-density SNP array and whole exome sequencing has increased the pace of gene discoveries and many new mutations are being published every month. The lack of uniform criteria has assigned multiple identifiers (or accession numbers) to the same MRT locus (e.g. MRT7 and MRT22). Here in this review we describe the molecular genetics of ARNSID, prioritize the candidate genes in uncharacterized loci, and propose a new nomenclature to reorganize the mutation data that will avoid the confusion of assigning duplicate accession numbers to the same ID locus and to make the data manageable in the future as well.

De novo hem- and lymphangiogenesis by endothelial progenitor and mesenchymal stem cells in immunocompetent mice.

Cellular pro-angiogenic therapies may be applicable for the treatment of peripheral vascular diseases. Interactions between mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) may provide such a treatment option. With the exception of some studies in man, experiments have only been performed in immunodeficient mice and rats. We studied an immunocompetent syngeneic mouse model. We isolated MSCs from bone marrow and EPCs from the lung of adult C57/Bl.6 mice and co-injected them in Matrigel subcutaneously in adult C57/Bl.6 mice. We demonstrate development of both blood vessels and lymphatics. Grafted EPCs integrated into the lining of the two vessel types, whereas MSCs usually did not incorporate into the vessel wall. Injections of each separate cell type did not, or hardly, reveal de novo angiogenesis. The release of VEGF-A by MSCs has been shown before, but its inhibitors, e.g., soluble VEGF receptors, have not been studied. We performed qualitative and quantitative studies of the proteins released by EPCs, MSCs, and cocultures of the cells. Despite the secretion of VEGF inhibitors (sVEGFR-1, sVEGFR-2) by EPCs, VEGF-A was secreted by MSCs at bioavailable amounts (350 pg/ml). We confirm the secretion of PlGF, FGF-1, MCP-1, and PDGFs by EPCs/MSCs and suggest functions for VEGF-B, amphiregulin, fractalkine, CXCL10, and CXCL16 during MSC-induced hem- and lymphangiogenesis. We assume that lymphangiogenesis is induced indirectly by growth factors from immigrating leukocytes, which we found in close association with the lymphatic networks. Inflammatory responses to the cellular markers GFP and cell-tracker red (CMPTX) used for tracing of EPCs or MSCs were not observed. Our studies demonstrate the feasibility of pro-angiogenic/lymphangiogenic therapies in immunocompetent animals and indicate new MSC/EPC-derived angiogenic factors.

Highly biocompatible behaviour and slow degradation of a LDH (layered double hydroxide)-coating on implants in the middle ear of rabbits.

Chronic inflammation can irreversibly damage components of the ossicular chain which may lead to sound conduction deafness. The replacement of impaired ossicles with prostheses does not reduce the risk of bacterial infections which may lead to loss of function of the implant and consequently to additional damage of the connected structures such as inner ear, meninges and brain. Therefore, implants that could do both, reconstruct the sound conduction and in addition provide antibacterial protection are of high interest for ear surgery. Layered double hydroxides (LDHs) are promising novel biomaterials that have previously been used as an antibiotic-releasing implant coating to curb bacterial infections in the middle ear. However, animal studies of LDHs are scarce and there exist only few additional data on the biocompatibility and hardly any on the biodegradation of these compounds. In this study, middle ear prostheses were coated with an LDH compound, using suspensions of nanoparticles of an LDH containing Mg and Al as well as carbonate ions. These coatings were characterized and implanted into the middle ear of healthy rabbits for 10 days. Analysis of the explanted prostheses showed only little signs of degradation. A stable health constitution was observed throughout the whole experiment in every animal. The results show that LDH-based implant coatings are biocompatible and dissolve only slowly in the middle ear. They, therefore, appear as promising materials for the construction of controlled drug delivery vehicles.

Development and evaluation of methotrexate-loaded nanoemulsion formulation for topical treatment of psoriasis.

Psoriasis is a chronic inflammatory disease that is becoming widespread and is associated with many kinds of additional severe diseases. The present study aimed to develop a methotrexate-loaded almond oil-based nanoemulsion formulation (MTX NE) for topical administration. The drug-loaded nanoemulsion formulation was prepared by high shear homogenization technique. The formulation's stability, as well as other physical and chemical characteristics, including entrapment effectiveness, drug release kinetics, skin permeability, skin irritation, and in vivo evaluation of the optimized formulation, was assessed. Additionally, imiquimod-induced psoriasis in rats was employed to investigate the efficacy of MTX NE against skin disorders. The MTX NE formulation was developed with a particle size of 18.74 ± 9.748 nm, a polydispersity index (PDI) of 0.198 ± 0.01, and an average entrapment efficiency of 79.65 ± 3.84%. The release kinetics model estimates 81.08% drug release at pH 5.5 after 24 h. The major layers of the skin, the epidermis, and dermis were successfully fluidized by the optimized MTX NE formulation, as shown by FTIR results, most likely enhancing drug retention and permeability. However, since Tween 80 and PEG 400 are well-known penetration enhancers, their application greatly accelerates these effects. Permeation data indicate that after 24 h, methotrexate was released from the nano-emulsion at 76.83 ± 4.98 g/cm2 with a flux rate of 2.385 ± 0.61 µg/cm2/h. The in vivo study conducted on rabbit skin showed that the enhanced skin penetration of the prepared MTX-loaded nanoemulsion formulation does not cause any structural modifications in the inter-cellular lipid layers of the stratum corneum. Rabbits used in the in vivo anti-psoriatic investigation demonstrated that MTX NE produced a 95% reduction in PASI. The pharmacokinetic profile revealed that the Cmax, Tmax, and t1/2 values were 8.63 µg/mL, 12.5 h, and 17.77 ± 2.21 h, respectively. These findings suggest that the formulation MTX NE is effective in treating psoriasis and may reduce psoriasis symptoms.

Layered double hydroxides as efficient drug delivery system of ciprofloxacin in the middle ear: an animal study in rabbits.

Chronic otitis media is a common disease often accompanied by recurrent bacterial infections. These may lead to the destruction of the middle ear bones such that prostheses have to be implanted to restore sound transmission. Surface coatings with layered double hydroxides (LDHs) are evaluated here as a possibility for drug delivery systems with convenient advantages such as low cytotoxicity and easy synthesis. Male New Zealand White rabbits were implanted with Bioverit(®) II middle ear prostheses coated with the LDH Mg(4)Al(2)(OH)(12)(SO(4))(2)·6H(2)O impregnated with ciprofloxacin. 12 (group 1) were directly infected with Pseudomonas aeruginosa and another 12 (group 2) 1 week after the implantation. Clinical outcome, blood counts, histological analyses and microbiological examination showed an excellent antimicrobial activity for group 1, whereas this effect was attenuated in animals where infection was performed 1 week after implantation. This is the first study to demonstrate an efficient drug delivery system with an LDH coating on prostheses in the middle ear.

A pH-responsive bi-MIL-88B MOF coated with folic acid-conjugated chitosan as a promising nanocarrier for targeted drug delivery of 5-Fluorouracil.

Cancer has remained one of the leading causes of death worldwide, with a lack of effective treatment. The intrinsic shortcomings of conventional therapeutics regarding tumor specificity and non-specific toxicity prompt us to look for alternative therapeutics to mitigate these limitations. In this regard, we developed multifunctional bimetallic (FeCo) bi-MIL-88B-FC MOFs modified with folic acid-conjugated chitosan (FC) as drug delivery systems (DDS) for targeted delivery of 5-Fluorouracil (5-FU). The bi-MIL-88B nanocarriers were characterized through various techniques, including powder X-ray diffraction, scanning electron microscopy, energy-dispersive X-ray, thermogravimetric analysis, and Fourier transform infrared spectroscopy. Interestingly, 5-FU@bi-MIL-88B-FC showed slower release of 5-FU due to a gated effect phenomenon endowed by FC surface coating compared to un-modified 5-FU@bi-MIL-88B. The pH-responsive drug release was observed, with 58% of the loaded 5-FU released in cancer cells mimicking pH (5.2) compared to only 24.9% released under physiological pH (5.4). The in vitro cytotoxicity and cellular internalization experiments revealed the superiority of 5-FU@bi-MIL-88B-FC as a highly potent targeted DDS against folate receptor (FR) positive SW480 cancer cells. Moreover, due to the presence of Fe and Co in the structure, bi-MIL-88B exhibited peroxidase-like activity for chemodynamic therapy. Based on the results, 5-FU@bi-MIL-88B-FC could serve as promising candidate for smart DDS by sustained drug release and selective targeting.

Collaterally Sensitive ß-Lactam Drugs as an Effective Therapy against the Pre-Existing Methicillin Resistant Staphylococcus aureus (MRSA) Biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is among the leading causes of nosocomial infections and forms biofilms, which are difficult to eradicate because of their increasing resistance to antimicrobial agents. This is especially true for pre-existing biofilms. The current study focused on evaluating the efficacy of three ß-lactam drugs, meropenem, piperacillin, and tazobactam, alone and in combination against the MRSA biofilms. When used individually, none of the drugs exhibited significant antibacterial activity against MRSA in a planktonic state. At the same time, the combination of meropenem, piperacillin, and tazobactam showed a 41.7 and 41.3% reduction in planktonic bacterial cell growth, respectively. These drugs were further assessed for biofilm inhibition and removal. The combination of meropenem, piperacillin, and tazobactam caused 44.3% biofilm inhibition, while the rest of the combinations did not show any significant effects. Results also revealed that piperacillin and tazobactam exhibited the best synergy against the pre-formed biofilm of MRSA, with 46% removal. However, adding meropenem to the piperacillin and tazobactam combination showed a slightly reduced activity towards the pre-formed biofilm of MRSA and removed 38.7% of it. Although the mechanism of synergism is not fully understood, our findings suggest that these three ß-lactam drugs can be used in combination as very effective therapeutic agents for the treatment of pre-existing MRSA biofilms. The in vivo experiments on the antibiofilm activity of these drugs will pave the way for applying such synergistic combinations to clinics.

The Global, Regional, and National Burden of Adult Lip, Oral, and Pharyngeal Cancer in 204 Countries and Territories: A Systematic Analysis for the Global Burden of Disease Study 2019.

Importance: Lip, oral, and pharyngeal cancers are important contributors to cancer burden worldwide, and a comprehensive evaluation of their burden globally, regionally, and nationally is crucial for effective policy planning. Objective: To analyze the total and risk-attributable burden of lip and oral cavity cancer (LOC) and other pharyngeal cancer (OPC) for 204 countries and territories and by Socio-demographic Index (SDI) using 2019 Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study estimates. Evidence Review: The incidence, mortality, and disability-adjusted life years (DALYs) due to LOC and OPC from 1990 to 2019 were estimated using GBD 2019 methods. The GBD 2019 comparative risk assessment framework was used to estimate the proportion of deaths and DALYs for LOC and OPC attributable to smoking, tobacco, and alcohol consumption in 2019. Findings: In 2019, 370 000 (95% uncertainty interval [UI], 338 000-401 000) cases and 199 000 (95% UI, 181 000-217 000) deaths for LOC and 167 000 (95% UI, 153 000-180 000) cases and 114 000 (95% UI, 103 000-126 000) deaths for OPC were estimated to occur globally, contributing 5.5 million (95% UI, 5.0-6.0 million) and 3.2 million (95% UI, 2.9-3.6 million) DALYs, respectively. From 1990 to 2019, low-middle and low SDI regions consistently showed the highest age-standardized mortality rates due to LOC and OPC, while the high SDI strata exhibited age-standardized incidence rates decreasing for LOC and increasing for OPC. Globally in 2019, smoking had the greatest contribution to risk-attributable OPC deaths for both sexes (55.8% [95% UI, 49.2%-62.0%] of all OPC deaths in male individuals and 17.4% [95% UI, 13.8%-21.2%] of all OPC deaths in female individuals). Smoking and alcohol both contributed to substantial LOC deaths globally among male individuals (42.3% [95% UI, 35.2%-48.6%] and 40.2% [95% UI, 33.3%-46.8%] of all risk-attributable cancer deaths, respectively), while chewing tobacco contributed to the greatest attributable LOC deaths among female individuals (27.6% [95% UI, 21.5%-33.8%]), driven by high risk-attributable burden in South and Southeast Asia. Conclusions and Relevance: In this systematic analysis, disparities in LOC and OPC burden existed across the SDI spectrum, and a considerable percentage of burden was attributable to tobacco and alcohol use. These estimates can contribute to an understanding of the distribution and disparities in LOC and OPC burden globally and support cancer control planning efforts.

Programmable Drug Release from a Dual-Stimuli Responsive Magnetic Metal-Organic Framework.

Along with the increasing incidence of cancer and drawbacks of traditional drug delivery systems (DDSs), developing novel nanocarriers for sustained targeted-drug release has become urgent. In this regard, metal-organic frameworks (MOFs) have emerged as potential candidates due to their structural flexibility, defined porosity, lower toxicity, and biodegradability. Herein, a FeMn-based ferromagnetic MOF was synthesized from a preassembled Fe2Mn(µ3-O) cluster. The introduction of the Mn provided the ferromagnetic character to FeMn-MIL-88B. 5-Fluoruracil (5-FU) was encapsulated as a model drug in the MOFs, and its pH and H2S dual-stimuli responsive controlled release was realized. FeMn-MIL-88B presented a higher 5-FU loading capacity of 43.8 wt % and rapid drug release behavior in a tumor microenvironment (TME) simulated medium. The carriers can rapidly release loaded drug of 70% and 26% in PBS solution (pH = 5.4) and NaHS solution (500 µM) within 24 h. The application of mathematical release models indicated 5-FU release from carriers can be precisely fitted to the first-order, second-order, and Higuchi models of release. Moreover, the cytotoxicity profile of the carrier against human embryonic kidney cells (HEK293T) suggests no adverse effects up to 100 µg/mL. The lesser toxic effect on cell viability can be attributed to the low toxicity values [LD50 (Fe) = 30 g·kg-1, (Mn) = 1.5 g·kg-1, and (terephthalic acid) = 5 g·kg-1] of the MOFs structural components. Together with dual-stimuli responsiveness, ferromagnetic nature, and low toxicity, FeMn-MIL-88B MOFs can emerge as promising carriers for drug delivery applications.

Define the Two Molecular Subtypes of Epithelioid Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is a fatal disease of respiratory system. Despite the availability of invasive biomarkers with promising results, there are still significant diagnostic and therapeutic challenges in the treatment of MPM. One of three main mesothelioma cell types, epithelioid mesothelioma makes up approximately 70% of all mesothelioma cases. Different observational findings are under process, but the molecular heterogeneity and pathogenesis of epithelioid malignant pleural mesothelioma (eMPM) are still not well understood. Through molecular analysis, expression profiling data were used to determine the possibility and optimal number of eMPM molecular subtypes. Next, clinicopathological characteristics and different molecular pathways of each subtype were analyzed to prospect the clinical applications and advanced mechanisms of eMPM. In this study, we identified two distinct epithelioid malignant pleural mesothelioma subtypes with distinct gene expression patterns. Subtype I eMPMs were involved in steroid hormone biosynthesis, porphyrin and chlorophyll metabolism, and drug metabolism, while subtype II eMPMs were involved in rational metabolism, tyrosine metabolism, and chemical carcinogenesis pathways. Additionally, we identified potential subtype-specific therapeutic targets, including CCNE1, EPHA3, RNF43, ROS1, and RSPO2 for subtype I and CDKN2A and RET for subtype II. Considering the need for potent diagnostic and therapeutic biomarkers for eMPM, we are anticipating that our findings will help both in exploring underlying mechanisms in the development of eMPM and in designing targeted therapy for eMPM.

Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels.

BACKGROUND: Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. RESULTS: In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels.Mouse lung microvascular endothelial cells (MLMVECs) were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony). These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs) in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. CONCLUSION: The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

A Participatory Investigation of Bovine Health and Production Issues in Pakistan.

Systems to record the frequency of animal health events in Pakistan are limited. A participatory approach was used to address gaps in farmers' knowledge and understanding of bovine health and production issues in five agroecological zones (AEZs) of Pakistan. Participatory tools, including simple ranking, pairwise ranking, constraint impact scoring, and constraint profiling were used in group discussions with farmers and animal health professionals (AHPs) in six districts of two provinces, Punjab and Sindh. The results of the ranking activities showed that foot-and-mouth disease (FMD), clinical mastitis, ticks, hemorrhagic septicemia, reproductive disorders, blackleg, and endoparasites were the most important bovine health and production constraints for small-scale dairy farmers. Constraint impact scoring showed that the participants perceived that: (1) milk production was severely affected by FMD and mastitis; (2) blackleg and parasitism led to poor growth rates and reduced meat production; (3) reproductive disorders and mastitis caused major economic losses (due to the high cost of treatment); and (4) blackleg and hemorrhagic septicemia were the leading causes of mortality in cattle and buffaloes. Although there was strong agreement in responses and constraint impact scores between farmers and AHPs, farmers were more concerned about health issues that cause high mortalities, whereas AHPs emphasized the importance of disorders with a high economic impact. Despite socioeconomic differences among AEZs, farmers' knowledge about bovine health and production constraints was similar. The findings from this study revealed that farmers had limited understanding of the risk factors and routes of transmission of various infectious diseases of bovines, which emphasizes the need to develop and implement tailored extension programs in Pakistan to control contagious diseases of animals and to improve the profitability of small-scale dairy farmers.

Molecular docking, anti-proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway.

Worldwide, cancer is still an area with high unmet medical need. Lead optimization efforts towards structure-based drug design were employed to discover newly synthesized hetero-steroid derivatives with promising anticancer effects against hepatocellular carcinoma (HCC). The aim of our study is to evaluate the anti-proliferative activity and the mechanism, a dual PI3K/mTOR inhibitor, and mechanism of action of a series of heterocylic androstane derivatives as anti-HCC agent. The cytotoxic effects of different heterocylic androstanes and 5FU as single agents, were assessed against both HepG2 cells and Non-malignant MDCK cell line to assess the toxicity. Then the underlying mechanism of compound 4 as most promising compound was evaluated using molecular docking, MTT assay, cell cycle analysis, DNA fragmentation, and real-time PCR. The results of MTT assay showed potential cytotoxic effect for compound 4 and 5 against liver cancer cell line with IC50 value 39.81 and 57.54 µM, respectively. Inhibition of the PI3K/AKT/mTOR pathway was achieved by compound 4, which was documented by molecular docking and augmented by gene expression analysis. Detailed mechanism revealed that compound 4 induced cell cycle arrest, DNA fragmentation, and induction of apoptosis by inhibition of anti-apoptotic genes, and upregulation of apoptotic genes. Our results shed a light on aminopyrazoloandrostane derivative 4 as an inhibitor of the PI3K/AKT/mTOR pathway, which might be acting as promising anti-liver cancer agent. Our data support further investigation of agents targeting the PI3K/AKT/mTOR.

Detection of rifampicin resistance of Mycobacterium tuberculosis using multiplex allele specific polymerase chain reaction (MAS-PCR) in Pakistan.

Drug resistance in tuberculosis (TB) is a major public health challenge in developing countries such as Pakistan. Multiplex allele specific polymerase chain reaction (MAS-PCR) is a DNA amplification method that could contribute to rapid detection and control of drug resistant tuberculosis (DR-TB) in Pakistan. The purpose of this study was to test the utility of MAS-PCR to detect resistance in Pakistan. Drug susceptibility testing (DST) was used to identify rifampicin resistant and susceptible clinical isolates from TB cases in Pakistan. MAS-PCR was used to detect the most frequent mutations in the gene rpoB among 213 resistant and 37 susceptible isolates. Among 213 clinical isolates, MAS-PCR identified mutation D435Y (Asp435Tyr) in 24 (11.3%) cases, H445Y (His445Tyr) in 14 (6.6%), S450L (Ser450Leu) in 124 (58.2%) and S450W (Ser450Trp) in 18 (8.4%) cases. MAS-PCR did not detect known mutations in 33 (15.5%) cases. Among 12 cases, a novel mutation at codon 434 (Met434Ile) and a common variant at codon 435 (Asp435Tyr) was detected in rpoB gene which is indicative of double mutation. In 4 isolates, a novel mutation at codon 432 (Gln432Pro) was identified. In an additional 4 isolates, mutations Met434Val and His445Asn were identified. Moreover, a mutation in rpoB (Leu452Pro) was found in 5 isolates. DNA sequencing confirmed the absence of mutations in rpoB in the 8 remaining isolates. MAS-PCR had 88.3% sensitivity and 100% specificity using DST as the reference, which suggested that this method could be implemented as an initial marker for screening of multi-drug resistant tuberculosis (MDR-TB) in Pakistan.

Exome sequence analysis in consanguineous Pakistani families inheriting Bardet-Biedle syndrome determined founder effect of mutation c.299delC (p.Ser100Leufs*24) in BBS9 gene.

BACKGROUND: Bardet-Biedl syndrome (BBS) is characterized by a heterogeneous phenotypic spectrum of retinopathy, intellectual disability (ID), obesity, polydactyly, and kidney dysfunctions as the major clinical features. Genetic investigations have reported 21 BBS genes, the products of which are mostly located at the centrosome, basal body or the ciliary transition zone. METHODS: In the present genetic report, we analyzed two apparently unrelated consanguineous BBS families from Dera Ismail Khan (D.I.Khan) district, Pakistan. Genetic mapping was performed using Whole exome sequencing and Sanger sequencing. RESULTS: Whole exome sequencing identified a recently reported single base deletion NM_001033604.1:c.299delC in the fourth exon of BBS9 in both families. The identified frameshift mutation is predicted to cause premature truncation of the expressed protein (p.Ser100Leufs*24). This mutation has previously been mapped in a consanguineous Pakistani family; therefore this is the second report of this particular mutation in two additional BBS families originating from different locations. CONCLUSION: We speculate the evolutionary significance of this mutation and assume its strong founder effect in the Khaisoori tribe of D.I.Khan. Based on these findings, we suggest developing a molecular diagnostic test that may be used for premarital and prenatal screening of families at risk of BBS.

Molecular genetic analysis of consanguineous families with primary microcephaly identified pathogenic variants in the ASPM gene.

Autosomal recessive primary microcephaly is a rare genetic disorder that is characterized by reduced head circumference and a varying degree of intellectual disability. Genetic studies on consanguineous families with primary microcephaly have identified 15 (MCPH) causative genes that include MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, CENPE, SASS6 MFSD2A ANKLE2 and CIT (Khan et al. 2014; Yamamoto et al. 2014; Alakbarzade et al. 2015;Morris-Rosendahl and Kaindl 2015; Basit et al. 2016). Physiologically, most of these MCPH proteins are involved in cell cycle and its regulation. In the present clinical genetic study, we have present two consanguineous Pakistani families segregating primary microcephaly and intellectual disability. These families were ascertained from the Saraiki ethnic part of Khyber-Pakhtunkhwa province in Pakistan. Whole exome sequencing in one family revealed a novel 1-bp deletion NM_018136.4: c.10013delA (p.Asp3338Valfs*2), while the other family showed a previously reported nonsense mutation NM_018136.4: c.9730C>T (rs199422195 (p.Arg3244*)) in ASPM gene. The novel frame-shift mutation (p.Asp3338Valfs*2) in ASPM presumably truncates the protein synthesis that results in loss of armadillo-type fold domain.

Controlled drug release from antibiotic-loaded layered double hydroxide coatings on porous titanium implants in a mouse model.

As an alternative to degradable organic coatings the possibility of using layered double hydroxides (LDHs) to generate implant coatings for controlled drug delivery was evaluated in vivo and in vitro. Coatings prepared from LDH suspensions dissolved slowly and appeared compatible with cultured cells. LDH coatings loaded with an antibiotic resulted in antibacterial effects in vitro. The LDH coating prolonged the drug release period and improved the proliferation of adherent cells in comparison to pure drug coatings. However, during incubation in physiological solutions the LDH coatings became brittle and pieces occasionally detached from the surface. For stress protection porous titanium implants were investigated as a substrate for the coatings. The pores prevented premature detachment of the coatings. To evaluate the coated porous implants in vivo a mouse model was established. To monitor bacterial infection of implants noninvasive in vivo imaging was used to monitor luminescently labeled Pseudomonas aeruginosa. In this model porous implants with antibiotic-loaded LDH coatings could antagonize bacterial infections for over 1 week. The findings provide evidence that delayed drug delivery from LDH coatings could be feasible in combination with structured implant surfaces.