Prospective exploratory evaluation of Cepheid Xpert Mycobacterium tuberculosis host response cartridge: a focus on adolescents and young adults.

BACKGROUND: An accurate, rapid, non-sputum-based triage test for diagnosing tuberculosis (TB) is needed. METHODS: A prospective evaluation of the Xpert-MTB-HR cartridge, a prototype blood-based host-response mRNA signature assay, among individuals presenting with TB-like symptoms was performed in Pakistan and results were compared to three reference standards: Xpert MTB/RIF Ultra, bacteriological confirmation (Xpert MTB/RIF Ultra and/or culture positivity), and composite clinical diagnosis (clinician diagnosis, treatment initiation, Xpert MTB/RIF Ultra, and/or culture positivity). Analyses were conducted both for the entire study cohort and separately in the adolescent and young adult cohort (ages 10-24). RESULTS: A total of 497 participants, ages 6-83, returned valid Xpert-MTB-HR results. When a diagnostic threshold was set for a sensitivity of >90%, specificity was 32% (95%CI 28-37) when compared to Xpert MTB/RIF Ultra, 29% (95%CI 25-34) when compared to a bacteriological confirmation, and 22% (95%CI 18-26) when compared to a composite clinical diagnosis. However, when evaluating only the adolescent and young adult cohort with a diagnostic threshold set for sensitivity of >90%, specificity was 82% (95%CI 74-89) when compared to Xpert MTB/RIF Ultra, 84% (95%CI 75-90) when compared to a bacteriological confirmation, and 54% (95%CI 44-64) when compared to a composite clinical diagnosis. CONCLUSIONS: While the Xpert-MTB-HR does not meet World Health Organization minimum criteria in the general population, in our study it does meet the minimum sensitivity and specificity requirements for a non-sputum-based triage test among adolescents and young adults when compared to Xpert MTB/RIF Ultra or bacteriological confirmation.

Occurrence of fungal microbial contamination in drinking water of megacity of Karachi (Pakistan) and their physico-chemical control.

The water quality in Karachi (Pakistan) is uncertain due to the occurrence of fungi and other microorganisms. A total of twenty-five water samples were collected from public places, educational institutes, hospitals, water supply systems and surface water of the canal of Karachi (Pakistan). The different fungal species including Acremonium sp., Alternaria alternata, Aspergillus flavus, A. fumigatus, A. sulphureus, Cladosporium sp., Fusarium sp., Clonostachys (Gliocladium) sp., Macrophomina phaseolina, Mucor racemosus, Paecilomyces sp. Penicillium chrysogenum, P. citrinum, P. commune, P. expansum, Rhizoctonia sp. and Stachybotrys sp. were isolated from these drinking water samples. However, the bacteria, microalgae and some other microorganisms were present in low concentrations. The reason for fungi infection and production of mycotoxicity depends upon various factors and the availability of their nutrients in filtration plants. The major threats to human health are fungal mycotoxicity which is responsible for carcinogenic and other lethal diseases. Mostly, the genus Aspergillus was dominated and isolated with a maximum of 88-98% of occurrence in the different samples of drinking water by the direct plate-spread method. For the control of fungi, various Physico-chemical coagulation treatments were used, but Potassium alum, clay pot, and hot water treatment disinfected effectively 69-70% removal of the fungi and its spore or mycelia from the water. In addition, it is concluded that drinking water purifications such as chlorination, filtration and lime did not eliminate thermophilic fungal spores or mycelia including Penicillium, Paecilomyces and Mucor from the water.

Advancement in nanomaterials for environmental pollutants remediation: a systematic review on bibliometrics analysis, material types, synthesis pathways, and related mechanisms.

Environmental pollution is a major issue that requires effective solutions. Nanomaterials (NMs) have emerged as promising candidates for pollution remediation due to their unique properties. This review paper provides a systematic analysis of the potential of NMs for environmental pollution remediation compared to conventional techniques. It elaborates on several aspects, including conventional and advanced techniques for removing pollutants, classification of NMs (organic, inorganic, and composite base). The efficiency of NMs in remediation of pollutants depends on their dispersion and retention, with each type of NM having different advantages and disadvantages. Various synthesis pathways for NMs, including traditional synthesis (chemical and physical) and biological synthesis pathways, mechanisms of reaction for pollutants removal using NMs, such as adsorption, filtration, disinfection, photocatalysis, and oxidation, also are evaluated. Additionally, this review presents suggestions for future investigation strategies to improve the efficacy of NMs in environmental remediation. The research so far provides strong evidence that NMs could effectively remove contaminants and may be valuable assets for various industrial purposes. However, further research and development are necessary to fully realize this potential, such as exploring new synthesis pathways and improving the dispersion and retention of NMs in the environment. Furthermore, there is a need to compare the efficacy of different types of NMs for remediating specific pollutants. Overall, this review highlights the immense potential of NMs for mitigating environmental pollutants and calls for more research in this direction.

Compounding Education in US PharmD Curricula.

OBJECTIVE: To determine the extent of compounding education (CE) offered in United States (US) doctor of pharmacy curricula. METHODS: A 24-item survey instrument addressing various aspects of CE was developed and validated. An email containing the link to the survey instrument was shared with instructors of compounding at 122 of 141 accredited schools and colleges of pharmacy in the US. RESULTS: Of these, 112 schools and colleges responded, rendering a survey response rate of 91.8%. Survey results indicate that CE is offered to a similar extent either as a required standalone course or as integrated instruction as part of a standard course. Whereas 70.8% of programs reported mostly hands-on training in CE in their curricula, there were about 11% programs that mostly offered didactic instruction in CE. Dispersed systems and semisolid formulations are the most prepared in nonsterile compounding, while proper hand washing, garbing, and gloving are the most taught techniques in sterile compounding. Compounding education is delivered principally by pharmaceutics faculty (62.3%) compared to practice faculty (32.1%). CONCLUSION: The survey determined the extent to which CE is addressed across different schools and colleges of pharmacy in the US. Although some institutions lack minimal nonsterile or sterile compounding facilities, they may improve by modeling the established programs in the country. Leadership at pharmacy institutions may need to allocate funds for CE, and support faculty who instruct in compounding.

Challenges in αCD38-chimeric antigen receptor (CAR)-expressing natural killer (NK) cell-based immunotherapy in multiple myeloma: Harnessing the CD38dim phenotype of cytokine-stimulated NK cells as a strategy to prevent fratricide.

BACKGROUND AIMS: Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38dim phenotype occurring during long-term cytokine stimulation of primary NK cells. METHODS: Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays. RESULTS: We verified the functionality of the αCD38-CAR-NK cells against CD38+ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo. CONCLUSIONS: Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

What is really 'Long COVID'?

The previous acute respiratory diseases caused by viruses originating from China or the middle east (e.g., SARS, MERS) remained fast developing short diseases without major sequalae or any long-lasting complications. The new COVID-19, on the other hand, not only that it rapidly spread over the world, but some patients never fully recovered or even if they did, a few weeks later started to complain not only of shortness of breath, if any, but general weakness, muscle pains and 'brain fog', i.e., fuzzy memories. Thus, these signs and symptoms were eventually labelled 'long COVID', for which the most widely used definition is 'new signs and symptoms occurring 4-8 weeks after recovering from acute stage of COVID-19'. The other most frequent manifestations associated with long COVID include headache, loss of memory, smell and of hair, nausea, and vomiting. Thus, long COVID is not a simple disease, but complex disorder of several organ systems malfunctioning; hence, it is probably more appropriate to call this a syndrome. The pathogenesis of long COVID syndrome is poorly understood, but initial and persistent vascular endothelial injury that often triggers the formation of microthrombi that if dislodged as emboli, damage several organs, especially in the brain, heart and kidney, by creating microinfarcts. The other major contributory mechanistic factor is the persistent cytokine storm that may last longer in long COVID patients than in others, probably triggered by aggregates of SARS-Co-2 discovered recently in the adrenal cortex, kidney and brain. The prevalence of long COVID is relatively high, e.g., initially varied 3-30%, and recent data indicate that 2.5% of UK population suffers from this syndrome, while in the US 14.7% of acute COVID-19 patients continued to have symptoms longer than 2 months. Thus, the long COVID syndrome deserves to be further investigated, both from clinical and basic research perspectives.

Generation of NK cells with chimeric-switch receptors to overcome PD1-mediated inhibition in cancer immunotherapy.

Multiple myeloma (MM) is an incurable hematological cancer, in which immune checkpoint inhibition (ICI) with monoclonal antibodies (mAbs) has failed due to uncontrollable immune responses in combination therapies and lack of efficacy in monotherapies. Although NK cell-specific checkpoint targets such as NKG2A and KIRs are currently being evaluated in clinical trials, the clinical impact of NK cells on the PD1 cascade is less well understood compared to T cells. Furthermore, while NK cells have effector activity within the TME, under continuous ligand exposure, NK cell dysfunctionality may occur due to interaction of PD1 and its ligand PD-L1. Due to above-mentioned factors, we designed novel NK cell specific PD1-based chimeric switch receptors (PD1-CSR) by employing signaling domains of DAP10, DAP12 and CD3ζ to revert NK cell inhibition and retarget ICI. PD1-CSR modified NK cells showed increased degranulation, cytokine secretion and cytotoxicity upon recognition of PD-L1+ target cells. Additionally, PD1-CSR+ NK cells infiltrated and killed tumor spheroids. While primary NK cells (pNK), expressing native PD1, showed decreased degranulation and cytokine production against PD-L1+ target cells by twofold, PD1-CSR+ pNK cells demonstrated increased activity upon PD-L1+ target cell recognition and enhanced antibody-dependent cellular cytotoxicity. PD1-CSR+ pNK cells from patients with MM increased degranulation and cytokine expression against autologous CD138+PD-L1+ malignant plasma cells. Taken together, the present results demonstrate that PD1-CSR+ NK cells enhance and sustain potent anti-tumor activity in a PD-L1+ microenvironment and thus represent a promising strategy to advance adoptive NK cell-based immunotherapies toward PD-L1+ cancers.

Assessment of T Cell Receptor Complex Expression Kinetics in Natural Killer Cells.

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.

Readiness, Reception, and Performance of Students in a Communications Course Delivered Amid the Pandemic.

Objective. To examine pharmacy student readiness, reception, and performance in a communications course amid the COVID-19 pandemic. Methods. First-year pharmacy students (2020 cohort) enrolled in a professional communications course completed a pre- and post-course questionnaire indicating their readiness and changes in reception toward online learning during the pandemic. Student learning performance (midterm and final examination grades) at the end of the course was compared with that of a class which took the same course face-to-face on campus the previous year (2019 cohort). Results. Student preference for face-to-face instruction decreased (difference in means = -1.59; p <.05), while their comfort level for online learning increased (difference in means = +0.38, p <.05) by the end of the course. No appreciable changes in rapport development with the instructor were perceived by the end of the study compared to the beginning. Student learning performance for the online cohort did not differ significantly (p >.05) compared to that of the 2019 cohort. Conclusion. The study demonstrates that students were partly prepared for online learning with the remainder of their maturation to it occurring while the quarter progressed. Remote online learning did not seem to impact student learning (grades) in this communications course during the COVID-19 crisis. Looking past the pandemic, educators and leadership at pharmacy schools and colleges may reassuringly continue to sustain online instruction, where deemed necessary, in their didactic curricula.

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan.

INTRODUCTION: Rifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. METHOD: A total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. RESULT: Among the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. CONCLUSION: Mutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

Role of receptor tyrosine kinases mediated signal transduction pathways in tumor growth and angiogenesis-New insight and futuristic vision.

In the past two decades, significant progress has been made in the past two decades towards the understanding of the basic mechanisms underlying cancer growth and angiogenesis. In this context, receptor tyrosine kinases (RTKs) play a pivotal role in cell proliferation, differentiation, growth, motility, invasion, and angiogenesis, all of which contribute to tumor growth and progression. Mutations in RTKs lead to abnormal signal transductions in several pathways such as Ras-Raf, MEK-MAPK, PI3K-AKT and mTOR pathways, affecting a wide range of biological functions including cell proliferation, survival, migration and vascular permeability. Increasing evidence demonstrates that multiple kinases are involved in angiogenesis including RTKs such as vascular endothelial growth factor, platelet derived growth factor, epidermal growth factor, insulin-like growth factor-1, macrophage colony-stimulating factor, nerve growth factor, fibroblast growth factor, Hepatocyte Growth factor, Tie 1 & 2, Tek, Flt-3, Flt-4 and Eph receptors. Overactivation of RTKs and its downstream regulation is implicated in tumor initiation and angiogenesis, representing one of the hallmarks of cancer. This review discusses the role of RTKs, PI3K, and mTOR, their involvement, and their implication in pro-oncogenic cellular processes and angiogenesis with effective approaches and newly approved drugs to inhibit their unrestrained action.

Impact of performance status on overall survival in patients with relapsed and/or refractory multiple myeloma: Real-life outcomes of daratumumab treatment.

BACKGROUND: Little is reported on the real-life impact of daratumumab in relapsed and/or refractory multiple myeloma patients (RRMM). We analyzed a cohort of 156 patients who received daratumumab as a single agent concerning ECOG status, eGFR, cytogenetics, lines of prior treatment, and their impact on survival. RESULTS: Eighty-two (53%) patients were triple refractory, 54 (35%) patients were single or double refractory, and 20 (12%) patients were non-refractory. Following daratumumab treatment, the progression-free survival (PFS) in these groups was 7.2%, 11.4%, and 53% (P < .001), and overall survival (OS) was 34%, 73%, and 58% (P < .001) at 36 months, respectively. Poor ECOG, three lines of prior treatment, and triple refractoriness were all associated with inferior PFS and OS in a multivariate analysis including ECOG, high-risk chromosomal aberrations, refractoriness, number of treatment lines, and eGFR. CONCLUSION: Daratumumab remains an attractive treatment option, especially in patients with poor performance and increased frailty. Furthermore, our observations suggest that patients with ECOG 2 and 3 status require additional supportive and/or palliative therapies to compensate for a potentially effective but encompassing late-line therapy. In conclusion, further prospective studies are needed to elucidate the impact of ECOG 2 and 3 status in patients with RRMM.

Environmental exposure pathway analysis of trace elements and autism risk in Pakistani children population.

The pursuit of industrialization and urbanization in developing countries disrupt the fragile environment, resulting in biogeochemical extra-emission of the trace elements into human inhabitance causing serious health concerns. We aimed to determine the associations between Autism spectrum disorder (ASD) risk and exposure to trace elements (As, Zn, Ni, Pb, Hg, Cu, Cd, and Co), associations between the internal doses and environmental sources of these elements were also assessed. Genetic susceptibility to toxins was assessed through GSTT1 and GSTM1 null polymorphism analysis. Our results showed that lower BMI in children was significantly associated with ASD (p < 0.05, AOR = 0.86; 95% CI: 0.76, 0.98). As was significantly higher in both hair (p < 0.01, AOR = 18.29; 95% CI: 1.98, 169) and urine (p < 0.01, AOR = 1.04; 95% CI: 1.01, 1.06) samples from children with ASD; urinary Hg (p < 0.05, AOR = 2.90; 95% CI: 1.39, 6.07) and Pb (p < 0.05, AOR = 1.95; 95% CI: 1.01, 3.77) were also positively associated with ASD. Regarding the genetic susceptibility, Cu was significantly associated with GSTM1 positive genotype (p < 0.05, AOR = 1.05; 95% CI: 1.00, 1.10). Children inhabiting the urban areas exposed to significantly higher levels of studied trace elements. The Estimated Daily Intake (EDI) values highlighted that the different land use settings resulted in children's source specific exposure to studied trace elements. The exposure pathway analysis showed that the distal factors of land-use settings associated with children increased exposure risk for most of the investigated elements, noticeably As, Pb and Hg associated with ASD prevalence.

Caveolin-1 and dynamin-2 overexpression is associated with the progression of bladder cancer.

Caveolae-mediated endocytosis regulates cell adhesion and growth in an anchorage-dependent manner. Studies of the endocytic function of caveolae have suggested a wide-ranging list of cargoes, including a number of receptors and extracellular proteins, ligands and nutrients from the extracellular matrix. Disruption of the processes of caveolae-mediated endocytosis mediated by signaling proteins is critical to cellular integrity. Caveolin-1 and dynamin-2 are the 2 major proteins associated with endocytotic function. Mechanistically, dynamin-2 has a co-equal role with caveolin-1 in terms of caveolae-derived endosome formation. Recent studies have revealed the pathological outcomes associated with the dysregulation of caveolin-1 and dynamin-2 expression. Increased expression levels of the gene for caveolin, Cav-1, resulting in augmented cellular metastasis and invasion, have been demonstrated in various types of cancer, and overexpression of the gene for dynamin-2, DNM2, has been associated with tumorigenesis in cervical, pancreatic and lung cancer. An increased expression of Cav-1 and DNM2 is known to be associated with the invasive behavior of cancer cells, and with cancer progression. Furthermore, it has been previously demonstrated that, in caveolar assembly and caveolae mediated endocytosis, Cav-1 interacts directly with DNM2 during the processes. Altered expression of the 2 genes is critical for the normal function of the cell. The expression patterns of Cav-1 and DNM2 have been previously examined in bladder cancer cell lines, and were each demonstrated to be overexpressed. In the present study, the expression levels of these 2 genes in bladder cancer samples were quantified. The gene expression levels of Cav-1 and DNM2 were identified to be increased 8.88- and 8.62-fold, respectively, in tumors compared with the normal controls. Furthermore, high-grade tumors exhibited significantly increased expression levels of Cav-1 and DNM2 (both P<0.0001) compared with the low-grade tumors. In addition, compared with normal control samples, the expression of the 2 genes in tumor samples was observed to be highly significant (P<0.0001), with a marked positive correlation identified for the tumors (Pearson's correlation coefficient, r=0.80 for the tumor samples vs. r=0.32 in the normal control samples). Taken together, the results of the present study demonstrated that the overexpression of Cav-1 and DNM2 genes, and a determination of their correlation coefficients, may be a potential risk factor for bladder cancer, in addition to other clinical factors.

Increased expression levels of Syntaxin 1A and Synaptobrevin 2/Vesicle-Associated Membrane Protein-2 are associated with the progression of bladder cancer.

Gene expression is tightly regulated in time and space through a multitude of factors consisting of signaling molecules. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) are membrane proteins responsible for the intercellular trafficking of signals through endocytosis and exocytosis of vesicles. Altered expression of SNARE proteins in cellular communication is the major hallmark of cancer phenotypes as indicated in recent studies. SNAREs play an important role in maintaining cell growth and epithelial membrane permeability of the bladder and are not only involved in cancer progression but also metastatic cell invasion through SNARE-mediated trafficking. Synaptobrevin2/Vesicle associated membrane protein-2 (v-SNARE) and Syntaxin (t-SNARE) form a vesicular docking complex during endocytosis. Some earlier studies have shown a critical role of SNARE in colon, lungs, and breast cancer progression and metastasis. In this study, we analyzed the relative expression of the STX1A and VAMP2 (SYB2) for their possible association in the progression and metastasis of bladder cancer. The profiling of the genes showed a significant increase in STX1A and VAMP2 expression (p < 0.001) in high-grade tumor cells compared to normal and low-grade tumors. These findings suggest that elevated expression of STX1A and VAMP2 might have caused the abnormal progression and invasion of cancer cells leading to the transformation of cells into high-grade tumor in bladder cancer.

Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene.

Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low density lipoprotein receptor (LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly reduced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combination of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events.

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study.

BACKGROUND: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. METHODS: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. FINDINGS: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. INTERPRETATION: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. FUNDING: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

In silico and in vitro evaluation of two novel oximes (K378 and K727) in comparison to K-27 and pralidoxime against paraoxon-ethyl intoxication.

Organophosphate (OP) poisoning is a major global health issue; while compounds from this group have been used intensively over the last century, an effective antidote is still lacking. Oxime-type acetylcholinesterase (AChE) reactivators are used to reactivate the OP inhibited AChE. Pralidoxime is the only US Food and Drug Administration approved oxime for therapeutic use but its efficacy has been disappointing. Two novel oximes (K378 and K727) were investigated in silico and in vitro and compared with an experimental oxime (kamiloxime; K-27) and pralidoxime. In silico the molecular interactions between AChE and oximes were examined and binding energies were assessed. LogP (predicted log of the octanol/water partition coefficient) was estimated. In vitro the intrinsic ability of the oximes to inhibit AChE (IC50) and their reactivation potency (R50) when used in paraoxon inhibited human RBC-AChE was determined. Molecular docking revealed that K378 and K727 bind to the peripheral site(s) with high binding energies in contrast to the central binding of K-27 and pralidoxime. LogP values indicating that the novel compounds are significantly less hydrophilic than K-27 or pralidoxime. IC50 of K378 and K727 were comparable (0.9 and 1 µM, respectively) but orders of magnitude lower than comparators. R50 values revealed their inability to reactivate paraoxon inhibited AChE. It is concluded that the novel oximes K378 and K727 are unlikely to be clinically useful. The in silico and in vitro studies described allow avoidance of unnecessary in vivo animal work and contribute to the reduction of laboratory animal use.

TNF-α Genetic Predisposition and Higher Expression of Inflammatory Pathway Components in Keratoconus.

Purpose: To date keratoconus (KC) pathogenesis is undefined; however, the involvement of inflammatory pathways in disease development is becoming apparent. In the present study, we investigated the role of a promoter region polymorphism rs1800629 (-308G>A) in the inflammatory pathway component TNF-α and its effects on the expression of TNF-α and downstream molecules tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), and interleukin 6 (IL-6) in KC development. Methods: TNF-α promoter polymorphism rs1800629 (-308G>A), was genotyped in 257 sporadic KC patients and 253 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was performed to assess for the -308G>A genotypes. Quantitative polymerase chain reaction (qPCR) was carried out to compare the mRNA expression of TNF-α, TNFR1, TNFR2, RELA, and IL6 in the corneal tissues of 20 KC patients and 20 donor controls. Results: The -308G>A genotype GA was found to be significantly associated with KC development (dominant model [odds ratio (OR) = 6.67 (95% confidence interval [CI] = 4.28-10.42), P < 0.001]) and allele-A (OR = 4.30, 95%CI = 2.93-6.34, P < 0.001). TNF-α serum levels were significantly raised in patients with GA genotype (196.5 ± 69.5 pg/mL) compared to reference genotype GG (21.7 ± 8.2 pg/mL) (P < 0.0001). There was a significant overexpression of TNF-α (P = 0.002), TNFR2 (P = 0.0001), RELA (P = 0.0117), and IL6 (P = 0.0007) in the KC corneal tissues as compared to the control. Conclusions: The GA genotype of the TNF-α -308G>A polymorphism is a significant genetic risk factor for the pathogenesis of KC. Moreover, this single nucleotide polymorphism (SNP) was observed to be associated with deregulated expression of downstream molecules, thus further reinforcing the role of the inflammatory pathway components in the development of KC.

Stability study on an anti-cancer drug 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid (CLEFMA) using a stability-indicating HPLC method.

CLEFMA, 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid, is a new chemical entity with anti-cancer and anti-inflammatory activities. Here, we report its stability in solution against stress conditions of exposure to acid/base, light, oxidant, high temperature, and plasma. The identity of the degradation products was ascertained by mass and proton nuclear magnetic resonance spectroscopy. To facilitate this study, we developed and validated a reverse phase high performance liquid chromatography method for detection of CLEFMA and its degradation. The method was linear over a range of 1-100 µg/mL; the accuracy and precision were within acceptable limits; it was stability-indicating as it successfully separated cis-/trans-isomers of CLEFMA as well as its degradation product. The major degradation product was produced from amide hydrolysis at maleic acid functionality caused by an acidic buffer, oxidant (3% hydrogen peroxide), or temperature stress (40-60 °C). The log k-pH profile showed that CLEFMA was most stable at neutral pH. In accelerated stability study we found that the shelf-life (T90% ) of CLEFMA at 25 °C and 4 °C was 45 days and 220 days, respectively. Upon exposure to UV-light (365 nm), the normally prevalent trans-CLEFMA attained cis-configuration. This isomerization also involved the maleic acid moiety. CLEFMA was stable in plasma from which it could be efficiently extracted by an acetonitrile precipitation method. These results indicate that CLEFMA is sensitive to hydrolytic cleavage at its maleic acid moiety, and it is recommended that its samples should be stored under refrigerated and light-free conditions, and under inert environment.