Cell wall metabolite levels as a function of the growth phase in Staphylococcus aureus and library screening for synergistic combinations of FDA approved drugs against methicillin resistant S. aureus

Title from PDF of title page, viewed August 25, 2022Dissertation advisor: William GutheilVitaIncludes bibliographical references (pages 140-159)Dissertation (Ph.D)--Division of Pharmacology and Pharmaceutical Sciences, Department of Chemistry. University of Missouri--Kansas City, 2022Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methods for quantifying cell wall biosynthesis intermediates. These include LC-MS/MS methods for quantifying cytoplasmic UDP-linked peptidoglycan biosynthesis intermediates. Retention and separation of these polar intermediates on C18 media were significantly improved using N, N-dimethylhexylamine as an ion-pairing agent. We also have developed Marfey’s reagent-based LC-MS/MS methods to stereospecifically resolve D- and L- amino acids and the key D-alanine-D-alanine intermediate involved in CWB on a reverse-phase analytical column. In the first part of this study, we examined how CWB intermediates and energy-rich nucleotide (ATP, etc.) levels change over the whole growth curve of S. aureus, from senescence to logarithmic growth and back to senescence. We first extended our established ion-pairing agent-based LC-MS/MS method for detecting UDP-linked intermediates, including energy-rich nucleotides (ATP, etc.) (unpublished). In this study, a culture of Methicillin-resistant Staphylococcus aureus (MRSA) American Type Culture Collection (ATCC 43300) is grown to senescence for 48 hours in Muller Hinton (MH) broth. From senescence to the exponential phase, the levels of UDP-linked intermediates increase significantly, and the levels of energy-rich nucleotides are low during these phases of the growth curve. Hence, UDP-linked intermediates appear to be the high-priority biomolecules required for maintaining bacterial physiology, where nucleotides with high energy are rapidly assimilated by UDP-linked intermediates. Additionally, the accumulation of energy-rich nucleotides coincided with the decline in cell growth rates, suggesting a concomitant reduction in their utilization. Retention and separation of glucosamine-1-phosphate (GlcN-1-P) and glucosamine-6-phosphate (GlcN-6-P) on a reverse-phase column is not feasible due to their high polarity. The second part of this study tested and demonstrated several derivatization procedures to improve the retention of GlcN-1-P and GlcN-6-P on a reverse-phase column. Derivatization with isobutyric anhydride, hexanoic anhydride, octanoic anhydride, and decanoic anhydride combined with multichannel LC-MS/MS detection demonstrated resolution and quantification of these three analytes. MRSA, vancomycin-resistant enterococcus (VRE), and vancomycin-resistant enterococcus (VSE) extracts were prepared and derivatized in this study. The LC-MS/MS analysis of these samples demonstrated peaks of GlcN-1-P and alpha and beta anomers of GlcN-6-P that were clearly resolved on a reverse-phase analytical column. Therefore, we performed a high-throughput screening of FDA-approved compounds in the presence and absence of ceftobiprole, identifying intrinsically active agents and synergistic combinations with ceftobiprole. A checkerboard analysis was then performed on potential ceftobiprole synergistic agents. Strongly synergistic agents included β-lactamase resistant β-lactams (cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, and cefotaxime). There was also evidence of synergy between ceftobiprole and both meropenem and imipenem. Vancomycin, balofloxacin, and floxuridine showed weak synergy. A similar antagonistic effect of mupirocin on ceftobiprole was also observed in other studies showing that it led to β-lactam resistance. We also performed a high-throughput library screening of FDA-approved drugs in the presence and absence of floxuridine against MRSA with the aim of surveying the full scope of floxuridine antibacterial synergy. This study examined joint MIC determination of the FDA library hits in combination with floxuridine, a high-resolution method to depict the fractional inhibitory concentration of √2. This way, several strong synergistic compounds (FICmin ≤ 0.5) like imipenem and biapenem were identified. Moreover, many agents showed weak synergy (0.5 < FICmin ≤ 0.75), such as nafcillin, doripenem, meropenem, and moxalactam.Cell wall metabolite levels as a function of growth phase in S. aureus -- Perform LC-MS/MS-based method development and quantification of Glucosamine-1-Phosphate (GlcN-1-P) and Glucosamine 6-Phosphate (GlcN-6-P) which are involved in the early steps of cell wall biosynthesis -- Library screening for synergistic combinations of FDA-approved drugs with Ceftobiprole against Methicillin-resistant Staphylococcus Aureus -- A high-throughput library screening of FDA-approved drugs with Floxuridine against MRSA -- General conclusio

Development and validation of novel method for simultaneous estimation of Atovaquone and Mefloquine hydrochloride in bulk drug using RP-HPLC

Atovaquone and Mefloquine hydrochloride are well known anti-malarial drugs. Literature survey reveals that there was no method available for the selected drug combination. In this way, here an endeavour has been made to develop simple, precise, fast method for simultaneous estimation of atovaquone and mefloquine hydrochloride in bulk drug by using RP-HPLC method. The method was carried out by using gradient HPLC on C18 column using Shimadzu prominence LC 20 AD and mobile phase comprised of Methanol:ACN:Water in the ratio of 85:7.5:7.5 (pH 2.9 was adjusted with OPA). The method was performed with 10µl injection volume. The UV detection was done at 231nm.The retention times of atovaquone and mefloquine hydrochloride were 7.6 and 2.6 min respectively.  The proposed method was validated according to ICH guidelines. The validation parameters were linearity, accuracy, precision (inter-day, intra-day and repeatability) and robustness etc. Linearity was in the range of 80-120µg/ml for atovaquone and 40-60µg/ml for mefloquine hydrochloride. The percent recoveries of both drugs were 99.99-100% and 92.05-99.09%. This method is suitable for the routine analysis of atovaquone and mefloquine hydrochloride in bulk drugs either individually or in mixtur

Design, synthesis and anticonvulsant activity of some new 5,7-dibromoisatin semicarbazone derivatives

A series of 5,7-dibromoisatin semicarbazones have been synthesized in good yield, involving aryl urea and aryl semicarbazide formation. The structures of the synthesized compounds were confirmed on the basis of their spectral data. All the compounds were evaluated for anticonvulsant and CNS depressant activities. Anticonvulsant activity was determined after intraperitoneal (i.p.) administration to mice by maximal electroshock (MES) induced seizure method and minimal motor impairment was determined by rotarod test. A computational study was carried out for prediction of pharmacokinetic properties and making them potentially promising agents for the treatment of epilepsy. Compounds (Z)-1-(5,7-dibromo-2-oxoindolin-3-ylidene)-4-(4-chlorophenyl)semicarbazide (DH-05), (Z)-1-(5,7-dibromo-2-oxoindolin-3-ylidene)-4-(3-chloro-4-fluorophenyl)semicarbazide (DH-11) and (Z)-1-(5,7-dibromo-1-methyl-2-oxoindolin-3-ylidene)-4-(3-chloro-4-fluorophenyl)semicarbazide (DH-12) exhibited prominent anticonvulsant effect in the series with little or no neurotoxicity and little CNS depressant effect as compared to standard drug

miRNAs regulate SIRT1 expression during mouse embryonic stem cell differentiation and in adult mouse tissues

SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD+ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells

Work from home practices as corporate strategy- an integrative review

The Covid 19 pandemic led to major changes at the individual, organisational and institutional levels of policy, productive functions, and organising. During Covid 19 morbidity, public institutions enforced social isolation, mandatory self-isolation, quarantines, and administrative regulatory lockdowns, which led to a movement away from the physical, material world and into an all-consuming digital universe. With growing interest in work-from-home (WFH) opportunities, this article provides an integrative review of 107 papers. It comprises the bibliometric analysis and manual review of the articles, on the basis of which we present an elaborative discussion and agenda for future research. According to the analysis, WFH looks a tad of a double-edged sword in that it may have major but unintended repercussions for institutions, and organizations as well as hidden, positive as well as negative consequences for individuals/employees. One of the significant insight from our analysis was the absence of HR function's strategic or operational input or oversight during corporate WFH strategies. We suggest several theoretical frameworks for further developing, theorizing, and empirically testing various aspects of WFH. Further, we recognise that WFH is becoming increasingly visible as a result of the pandemic scenario and significant technical advancements, which must be reflected in the research. Finally, because WFH represents a significant disruption in how organizations produce work and manage it, we propose employee and managerial consequences as future research agendas. [Abstract copyright: © 2023 The Authors.

MicroRNAs modulate SARS-CoV-2 infection of primary human hepatocytes by regulating the entry factors ACE2 and TMPRSS2.

BACKGROUND AND AIMS Severe acute respiratory syndrome coronavirus (SARS-CoV-2) preferentially infects the respiratory tract; however, several studies have implicated a multi-organ involvement. Hepatic dysfunctions caused by SARS-CoV-2 infection have been increasingly recognized and described to correlate with disease severity. To elucidate molecular factors that could contribute towards hepatic infection, we concentrated on microRNAs (miRNAs), a class of small non-coding RNAs that modulate various cellular processes and which are reported to be differentially regulated during liver injury. We aimed to study the infection of primary human hepatocytes (PHH) with SARS-CoV-2 and to evaluate the potential of miRNAs for modulating viral infection. METHODS We analysed liver autopsies from a coronavirus disease 19 (COVID-19)-positive cohort for the presence of viral RNA using Nanopore sequencing. PHH were used for the infection with SARS-CoV-2. The candidate miRNAs targeting angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) were identified using in silico approaches. To discover the potential regulatory mechanism, transfection experiments, qRT-PCRs, western blots and luciferase reporter assays were performed. RESULTS We could detect SARS-CoV-2 RNA in COVID-19-positive liver autopsies. We show that PHH express ACE2 and TMPRSS2 and can be readily infected with SARS-CoV-2, resulting in robust replication. Transfection of selected miRNA mimics reduced SARS-CoV-2 receptor expression and SARS-CoV-2 burden in PHH. In silico and biochemical analyses supported a potential direct binding of miR-141-3p to the SARS-CoV-2 genome. CONCLUSION We confirm that PHH are susceptible to SARS-CoV-2 infection and demonstrate selected miRNAs targeting SARS-CoV-2 entry factors and/or the viral genome reduce viral loads. These data provide novel insights into hepatic susceptibility to SARS-CoV-2 and associated dysfunctions in COVID-19

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models

Application of frequency ratio, statistical index, and weights-of-evidence models and their comparison in landslide susceptibility mapping in Central Nepal Himalaya

The Mugling–Narayanghat road section falls within the Lesser Himalaya and Siwalik zones of Central Nepal Himalaya and is highly deformed by the presence of numerous faults and folds. Over the years, this road section and its surrounding area have experienced repeated landslide activities. For that reason, landslide susceptibility zonation is essential for roadside slope disaster management and for planning further development activities. The main goal of this study was to investigate the application of the frequency ratio (FR), statistical index (SI), and weights-of-evidence (WoE) approaches for landslide susceptibility mapping of this road section and its surrounding area. For this purpose, the input layers of the landslide conditioning factors were prepared in the first stage. A landslide inventory map was prepared using earlier reports, aerial photographs interpretation, and multiple field surveys. A total of 438 landslide locations were detected. Out these, 295 (67 %) landslides were randomly selected as training data for the modeling using FR, SI, and WoE models and the remaining 143 (33 %) were used for the validation purposes. The landslide conditioning factors considered for the study area are slope gradient, slope aspect, plan curvature, altitude, stream power index, topographic wetness index, lithology, land use, distance from faults, distance from rivers, and distance from highway. The results were validated using area under the curve (AUC) analysis. From the analysis, it is seen that the FR model with a success rate of 76.8 % and predictive accuracy of 75.4 % performs better than WoE (success rate, 75.6 %; predictive accuracy, 74.9 %) and SI (success rate, 75.5 %; predictive accuracy, 74.6 %) models. Overall, all the models showed almost similar results. The resultant susceptibility maps can be useful for general land use planning