IN SILICO LIGAND-BASED 2D PHARMACOPHORE GENERATION FOR H+/K+ ATPASE INHIBITORS

Objectives: At the beginning of the 20th century, Peptic Ulcer became the most prevalent disease as non-steroidal anti-inflammatory drugs (NSAIDs) proved ineffective. Researches proved proton pump inhibitors as most successful drugs for the treatment of peptic ulcer. Hence, a ligand based pharmacophore was generated on Ligand Scout based on fifteen selected H+/K+ ATPase inhibitors. Methods:  A pharmacophore model with three Hydrogen bond acceptors, one Hydrogen bond donor and one Hydrophobic Domain was developed. The distance between these features was estimated on Visual Molecular Dynamics (VMD) software. Results:  The range between HBA-HBD was found to be 1.89-2.96A. The range between HBD-HP was 4.00-5.46A and range between HP-HBA was 1.89-2.96A. Conclusion: Study concludes that the model that is designed has three Hydrogen bond acceptors, one Hydrogen bond donor and one hydrophobic domain. This research will thus help in designing new drugs for the treatment of peptic ulcer disease. Peer Review History: Received 9 September;   Revised 12 September; Accepted 13 September, Available online 15 September 2017 Academic Editor: Dr. Sally A. El-Zahaby, Pharos University in Alexandria, Egypt, [email protected] UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file:        Reviewer's Comments: Average Peer review marks at initial stage: 6.5/10 Average Peer review marks at publication stage: 8.5/10 Reviewer(s) detail: Dr. Kingsley C Anukam, University of Benin, Nigeria, [email protected] Dr.  Masoomeh S Ghahfarokhi, University of Benin, Nigeria, [email protected]

Caffeine – An Invisible Addiction

Caffeine, an alkaloid, is a stimulant of the central nervous system and is the most popularly consumed psychoactive drug worldwide. This stimulant can have both positive and negative effects on health. The objective of this study was hence to determine the common side effects associated with consumption of caffeinated drinks along with any symptoms stated by the participants that indicate withdrawal. A questionnaire-based descriptive epidemiological study was conducted at three major universities of Lahore, Pakistan. Among the target population that fell in the age ranging from 18-28 years, 35% started consuming caffeinated drinks between the ages 10-15, while tea was consumed by 60% of the sample population. 50% consume caffeinated drinks once or twice a day whereas 60% could refrain from consuming a single drink per day. 38.5% stated to suffer from increased urination while 22% suffered from increased blood pressure upon consumption. Caffeine addiction is one of the most prevalent and ever-expanding addictions in the world which must be either eliminated from diet or replaced by a healthier alternative

Oil based inactivated vaccine formulation for furunculosis (A. salmonicida) and protective immune response of rainbow trout and brown trout

The development and growth of the fisheries and aquaculture industries are significantly hampered by illnesses. It is critical to combat pathogenic illnesses, especially bacterial ones. Furunculosis in salmon is mostly brought on by Aeromonas salmonicida in rainbow and brown trout. To control this pathogen, vaccines have been identified as a significant tool. In the present study, we have formulated an inactivated vaccine with oil as an adjuvant and estimated its efficacy. The lethal dose of ArS-Pak-19, was calculated and injected intraperitoneally to the fishes. To analyze the infection, samples of kidney, liver, spleen, and blood were collected at specific times. To estimate the immunogenicity of the vaccine, an experiment was designed. One hundred sixty fishes were distributed into 8 tanks including, six experimental groups and two control groups with its replicates, vaccines injected intraperitoneally 1.6 × 107 , 1.6 × 108 , and 1.6 × 109 and blood samples were taken fortnightly for 56 days to calculate the antibodies titers. After immunization these groups were challenged with Aeromonas salmonicida (ArS-Pak-19) intraperitoneally. At 7th day of post infection, it appeared in the liver, spleen, and kidney. The relative percentage of survival was estimated with control groups at 30 days after challenge. The relative percentage of survival was 80%. The IgM titers were higher at 24 days of post immunization. We also analyzed that antibodies non-specifically bound with the A-layer of Aeromonas salmonicida. The findings of this study offer evidence that vaccinations boost fishes immunity and serve as a roadmap to further vaccination initiatives

Chemical Biology of Chromosome Segregation in Pseudomonas aeruginosa

Chromosome duplication and cell division are two critical events of a cell’s life cycle that occur with high precision across diverse environmental conditions. Inheriting a single copy of a chromosome by each daughter cell is essential for survival. An important aspect ensuring this is chromosome organization by condensins and other nucleoid-associated proteins, which, through varied levels of compaction, enables numerous DNA-dependent processes. Passing down a fully duplicated and condensed chromosome during cell division is not a trivial job for bacteria since they efficiently adapt to the constantly changing environments and conditions that may cause DNA damage or interrupt normal progression and coordination between cell cycle events. Unlike in eukaryotes, chromosome duplication and cell division occur concurrently. To ensure the coordinated progression of the cell cycle and viability of offspring, bacteria employ a variety of pathways, some of which are known, while others are still unknown or yet to be discovered, for coordinating DNA duplication with cell division. The role of global chromosome compaction in the process of DNA duplication and cell division also remains unknown. Therefore, a better understanding of these complex processes and, more importantly, the mechanism of proteins involved in these processes could significantly enhance our existing knowledge. This, in turn, can enable us to manipulate chromosomes for various applications, find new targets for drug discovery, and even develop complementary approaches to genetic manipulations to explore cellular mechanisms of these processes in bacteria. The current study investigated the role of two redundant systems, condensins and ParB, in the organization and assembly of replisome, segrosome, and divisome at the midcell and cell quarters in P. aeruginosa. P. aeruginosa is a Gram-negative, opportunistic human pathogen infecting immunocompromised patients and is the major cause of mortality and morbidity in patients suffering from cystic fibrosis. It has a reputation for being notoriously resistant to multiple antibiotics, making infections caused by P. aeruginosa life-threatening. Till now, the vast majority of studies have focused on multi-drug resistant aspects of P. aeruginosa, but lately, it has been gaining attention in the scientific world as an interesting model for cell cycle studies due to the discovery of new proteins and pathways associated with the cell cycle. Previously, our lab discovered a new class of condensins, MksB, a relative of MukB, that might have comparable functions in DNA compaction and segregation in P. aeruginosa and prompted us to further explore other life cycle facets of this fascinating yet deadly pathogen. Our lab also found that the condensins (SMC and MksB) and the ParABS system, mediating dynamics, and positioning of origin of replication (oriC) are synthetically lethal. Deletion of either affected oriC dynamics, global chromosome layout, and, most importantly, disrupted chromosome segregation process as evidenced by the production of anucleate cells. These results piqued our curiosity, and further investigations revealed that the condensins and the ParABS system have additional biological roles in the biogenesis of bacterial cells. Condensins and the ParABS system are presently thought to be involved in chromosome compaction and segregation. In the first project (Chapter 3), we investigated which proteins assembled and occupied the midcell and cell quarters. Next, we explored the sequence of events at the midcell and cell quarters during the cell cycle and proposed a mechanism for the biogenesis of new bacterial cells governed by condensins and ParB. Using fluorescence microscopy, we tracked the dynamics of key proteins involved in chromosome replication, segregation, and cell division relative to oriC. Our findings revealed that these processes are closely linked, with all three occurring at the midcell in newborn cells and later on at cell quarters. After characterizing and establishing the role of condensins and the ParABS system in chromosome duplication and cell division process, we next characterized and employed chemical probes to study the mechanism and role of condensins and ParB in these processes. P. aeruginosa, like many other Gram-negative bacteria, poses a significant in healthcare settings due to its ability to develop antibiotic resistance. The rise in multi-drug-resistant bacteria is swiftly diminishing the effectiveness of current antibiotics, resulting in high mortality and morbidity rates, and increased public health challenges due to bacterial infections. Since most of the drugs that are currently in late-stage clinical trials are analogs of the antibiotics towards which bacteria are rapidly becoming resistant, there is a dire need to develop or screen new antibacterials that are unique and mechanistically different from the existing ones. Based on the synthetic lethality of condensins (SMC and MksB) and ParB, we developed an assay to screen a library of fungal extracts for potential inhibitors of chromosome segregation. Specifically, we screened for compounds that inhibited the ParABS system (Chapter 4) and condensins (Chapter 5) based on their increased activity in condensin and ParB mutants, respectively. In the second project (Chapter 4), we report the discovery of a family of compounds extracted from Humicola sp. that had enhanced activity condensin mutants and were later characterized as the first known inhibitors of ParB protein. Specifically, we found new analogs of sterigmatocystin, one of which, 4-hydroxy-sterigmatocystin, 4HS, displayed an antibacterial activity and induced the phenotype typical for parAB mutants, including defects in chromosome segregation and cell division. The data describe an inhibitor of the ParAB pathway and expand the known spectrum of activities of sterigmatocystin to include bacterial chromosome segregation. Furthermore, 4HS was also found to disrupt oriC dynamics in a manner similar to ParB. In the third project (Chapter 5), we discovered compounds derived from Aspergillus sp. that were synergistic with the deletion of parB. These compounds inhibited cell growth and induced cell lysis in P. aeruginosa. The hit compounds were found to be analogs of previously reported asteltoxins that inhibited respiration. Our subsequent experiments confirmed that our hit compound, 176LJ22M, also inhibited membrane bioenergetics by suppressing ATP levels and dissipating proton motive force (PMF), leading to growth inhibition and cell lysis. Comparison with known inhibitors of respiration further revealed that 176LJ22M not only inhibits respiration but also targets chromosome segregation, as evidenced by the increased frequency of anucleate cells. Overall, in the current thesis, the fundamental concepts about the cell cycle and the associated proteins involved in its progression were first established. This was followed by the development of complementary approaches to genetic manipulations to explore the cellular mechanism of chromosome segregation in P. aeruginosa. Additionally, chemical probes were discovered that targeted key proteins involved in chromosome segregation, including condensins and the ParABS system. These probes enabled deeper insights into the roles of these proteins in the segregation of chromosomes during the cell cycle. Keywords: chromosome segregation, condensins, ParABS system, cell cycle, chemical probes, sterigmatocystin, asteltoxin, Pseudomonas aeruginos

Changing Antimicrobial Susceptibility and Resistance Pattern of Acinetobacter Species over the Last Eight Years in a Tertiary Care Hospital in Lahore, Pakistan

Background: Acinetobacter spp. is a highly resistant nosocomial pathogen that leads to a broad range of human infections resulting in high morbidity and mortality. Due to unpredictable MDR patterns of Acinetobacter spp., it is imperative to know the institutional prevalent susceptibility profiles of these residing pathogens. The objective of this study was to determine the antimicrobial susceptibility pattern of Acinetobacter species over the last 8 years in a tertiary care hospital in Lahore, Pakistan.Material and Methods: A retrospective study was carried out in Lahore General Hospital, a tertiary care hospital in Lahore, Pakistan. Eight-year data was gathered from January 2012 to December 2019. All specimens were handled according to standard operating procedures in the microbiology laboratory of the Pathology department of Lahore General Hospital. The Acinetobacter spp. were identified in the laboratory by Gram staining, oxidase test, catalase test and Triple sugar iron fermentation and their antibiotic sensitivity pattern was noted.Results: The highest yield of Acinetobacter spp. from the clinical specimen was isolated from pus followed by tracheal secretion, blood, and urine in the last three years (from 2017 to 2019). Most of the isolates were multi-drug resistant (MDR). There was a progressive increase in resistance of Acinetobacter spp. The highest progression in resistance was observed among the cephalosporin and quinolone group of antibiotics.Conclusions: Increased resistance to commonly used antimicrobials against Acinetobacter species has been observed with the highest resistance to quinolones and cephalosporins

In Silico Prediction of New Inhibitors for Kirsten Rat Sarcoma G12D Cancer Drug Target Using Machine Learning-Based Virtual Screening, Molecular Docking, and Molecular Dynamic Simulation Approaches

Single-point mutations in the Kirsten rat sarcoma (KRAS) viral proto-oncogene are the most common cause of human cancer. In humans, oncogenic KRAS mutations are responsible for about 30% of lung, pancreatic, and colon cancers. One of the predominant mutant KRAS G12D variants is responsible for pancreatic cancer and is an attractive drug target. At the time of writing, no Food and Drug Administration (FDA) approved drugs are available for the KRAS G12D mutant. So, there is a need to develop an effective drug for KRAS G12D. The process of finding new drugs is expensive and time-consuming. On the other hand, in silico drug designing methodologies are cost-effective and less time-consuming. Herein, we employed machine learning algorithms such as K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF) for the identification of new inhibitors against the KRAS G12D mutant. A total of 82 hits were predicted as active against the KRAS G12D mutant. The active hits were docked into the active site of the KRAS G12D mutant. Furthermore, to evaluate the stability of the compounds with a good docking score, the top two complexes and the standard complex (MRTX-1133) were subjected to 200 ns MD simulation. The top two hits revealed high stability as compared to the standard compound. The binding energy of the top two hits was good as compared to the standard compound. Our identified hits have the potential to inhibit the KRAS G12D mutation and can help combat cancer. To the best of our knowledge, this is the first study in which machine-learning-based virtual screening, molecular docking, and molecular dynamics simulation were carried out for the identification of new promising inhibitors for the KRAS G12D mutant

Ultra-Responses of Asphodelus tenuifolius L. (Wild Onion) and Convolvulus arvensis L. (Field Bindweed) against Shoot Extract of Trianthema portulacastrum L. (Horse Purslane)

Weed infestation is a prime challenge coupled with lowering crop production owing to their competition with crop plants for available resources such as nutrients, water, space, moisture, and sunlight. Among weed control methods, the implementation of synthetic herbicides offers an instant solution for getting rid of weeds; however, they are a direct source of potential hazards for humans and generate resistance against synthetic weedicides, making them less effective. Allelopathy is something that happens in nature that can be used as a weed control method that increases crop yield and decreases dependency on synthetic chemicals. The mode of action of some phytochemicals corresponds to synthetic herbicides. Due to this feature, allelochemicals are used as bio-herbicides in weed management and prove more environmentally friendly than synthetic weedicides. The present investigation aims to assess the ultra-responses of A. tenuifolius and C. arvensis, while growing them in a pot experiment. Various levels of shoot extract (L2, L3, and L4) of T. portulacastrum along with the L1 (distilled water) and L5 (synthetic herbicide) were applied to the weeds. Results indicated that aqueous extracts of shoot of T. portulacastrum significantly (p ≤ 0.05) affect all the measured traits of weeds and their effects were concentration specific. All morphological parameters were suppressed due to biotic stress with an increase in free amino acids and calcium ions along with a decline in metaxylem cell area and cortical thickness in the root, while the vascular bundle area increased. The shoot extract intrusive with metabolisms corresponded with the synthetic herbicide. It is concluded that Trianthema shoot extract has a powerful phytotoxic impact on weeds (A. tenuifolius and C. arvensis) and can be used in bio-herbicide production