Design and Synthesis of Benzimidazole Carboxamide Cysteine Protease Inhibitors as Promising Anti-leishmanial Agents.

INTRODUCTION: More than 20 protozoan species of Leishmania are responsible for causing Leishmaniasis, an infection spread by blood-feeding phlebotomine sandflies. A narrow pool of drugs is currently available rendering the current drug stratagem to treat this infection . Development of novel, less toxic, and more effective regimens is thus a need of the hour. Design and synthesis of benzo[d]imidazole carboxamides as agents to combat Leishmaniasis are also required. METHODS: 14 benzo[d]imidazole carboxamides were synthesized and gauged against L. donovani promastigotes and intramacrophage amastigote forms. All of the tested compounds exhibited significant anti-promastigote properties with IC50 well below 10 uM. Compounds 4a, 4b, and 4d, showing the highest anti-parasitic activity against promastigote forms (IC50 0.91- 1.33 µM), were also found to be associated with better anti-leishmanial potential (IC50 0.78- 1.67 µM) against the intramacrophage amastigotes comparable to Amphotericin-B (0.13 µM), a drug used for Leishmaniasis. Compound (4a), namely N-(2-(trifluoromethyl)-1Hbenzo[ d]imidazol-5-yl)benzo[d][1,3]-5-carboxamide-dioxole, was found to be most potent against L. donovani amastigotes among all the tested compounds, and demonstrated better antileishmanial properties (IC50 0.78 µM) when compared to the standard. Compound 4a was also assessed for its toxicity profile against THP-1 human monocytic cells. To establish the molecular target(s) in silico, molecular docking studies were performed against cysteine protease, a putative virulence factor of Leishmania parasites, and nucleoside diphosphate kinase, an enzyme with a critical role in nucleotide recycling, also associated with resistance in Leishmania strains. Compound 4a showed better binding affinity than the standard to these targets; furthermore, the molecular dynamic simulation studies further affirmed the stability of compound 4a, within the active site of the targets. In vitro, cysteine protease inhibitory activity (IC50 8.53 µM) using Bz-Arg-AMC hydrochloride fluorogenic peptide substrate established the promising potential of 4a as a cysteine protease inhibitor. RESULT: Computational ADMET analysis indicated appropriate pharmacokinetic profile and physicochemical characteristics for all members of the synthesized library. CONCLUSION: Both in vitro and in silico studies indicate that the synthesized imidazole carboxamides can act as potent hits and that N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5- yl)benzo[d][1,3]-5-carboxamide-dioxole 4a can be an effective hit molecule which can be further developed into potent lead molecule (s) to fight Leishmania donovani.

A supported primary health pathway for mild traumatic brain injury quality improvement report.

Introduction Best-practice guidelines recommend that patients are followed-up to check if they have recovered following a mild traumatic brain injury (mTBI) and to refer to concussion services, if needed. However, in New Zealand, rates of follow-up are low and access to concussion services can be delayed. Aim We aimed to improve rates of follow-up and access to concussion services for mTBI patients aged ≥8 years by the implementation of a supported health pathway and test its success. Methods The pathway included a decision support tool, funded follow-up appointments, clinician training and a patient education resource. Sociodemographics, injury details and proportions of patients receiving a follow-up by type and time were extracted from the Accident Compensation Corporation (ACC) database between 18 May 22 and 30 June 23 and compared to national ACC data prior to implementation. Results Data were extracted for 220 patients, with a mean age of 31.5 years, 51.4% female and 21.4% Maori and Pacific. There was an increase in the proportion of patients receiving a follow-up from 36% pre-implementation to 56.8% post-implementation. Sixty-three patients (28.6%) accessed a concussion service post-implementation compared to 10% pre-implementation. Time to concussion service reduced from an average of 55 (s.d. = 65.4) to 37 days (29.5). Discussion Risk factor criteria within the decision support tool need to be weighted to improve specificity of referrals. Timing from injury to medical review in primary care needs to be considered. This quality improvement project provides preliminary evidence for implementation of a supported health care pathway for mTBI.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Social Disadvantage and Multimorbidity Including Oral Conditions in the United States.

Existing studies on multimorbidity have largely excluded oral diseases in multimorbidity prevalence estimates. The reason behind this is somewhat unclear, as chronic oral conditions are highly prevalent, affecting over half the global population. To address this gap, we examined the relationship between social disadvantage and multimorbidity, stratifying by the inclusion and exclusion of oral conditions. For participants aged 30 y and over (n = 3,693), cross-sectional analysis was carried out using the US National Health and Nutrition Survey (2013-2014). Multimorbidity was defined as having 2 or more chronic conditions. Five medical conditions were examined: diabetes, asthma, arthritis, cardiovascular disease, and depression, as well as 4 oral health conditions: caries, periodontal disease, number of teeth, and edentulousness. Education and income poverty ratio were selected as measures of social disadvantage. Multimorbidity prevalence estimates according to social disadvantage were analyzed on an absolute and relative scale using inverse probability treatment weighting (IPTW), adjusting for age, sex, and ethnicity. The inclusion of oral health conditions in the assessment of multimorbidity increased the overall prevalence of multimorbidity from 20.8% to 53.4%. Findings from IPTW analysis demonstrated clear social gradients for multimorbidity estimates stratified by the exclusion of oral conditions. Upon inclusion of oral conditions, the prevalence of multimorbidity was higher across all social groups for both education and income. Stratifying by the inclusion of oral conditions, the mean probability of multimorbidity was 27% (95% confidence interval [CI], 23%-30%) higher in the low-education group compared to the high-education group. Similarly, the mean probability of multimorbidity was 44% (95% CI, 40%-48%) higher in the low-income group. On a relative scale, low education was associated with a 1.52 times (95% CI, 1.44-1.61) higher prevalence of multimorbidity compared to high education. Low income was associated with a 2.18 (95% CI, 1.99-2.39) higher prevalence of multimorbidity. This novel study strongly supports the impact of chronic oral conditions on multimorbidity prevalence estimates.

High-fidelity, low-cost synthetic training model for fetoscopic spina bifida repair.

BACKGROUND: Fetoscopic spina bifida repair is increasingly being practiced, but limited skill acquisition poses a barrier to widespread adoption. Extensive training in relevant models, including both ex vivo and in vivo models may help. To address this, a synthetic training model that is affordable, realistic, and that allows skill analysis would be useful. OBJECTIVE: This study aimed to create a high-fidelity model for training in the essential neurosurgical steps of fetoscopic spina bifida repair using synthetic materials. In addition, we aimed to obtain a cheap and easily reproducible model. STUDY DESIGN: We developed a 3-layered, silicon-based model that resemble the anatomic layers of a typical myelomeningocele lesion. It allows for filling of the cyst with fluid and conducting a water tightness test after repair. A compliant silicon ball mimics the uterine cavity and is fixed to a solid 3-dimensional printed base. The fetal back with the lesion (single-use) is placed inside the uterine ball, which is reusable and repairable to allow for practicing port insertion and fixation multiple times. Following cannula insertion, the uterus is insufflated and a clinical fetoscopic or robotic or prototype instruments can be used. Three skilled endoscopic surgeons each did 6 simulated fetoscopic repairs using the surgical steps of an open repair. The primary outcome was surgical success, which was determined by water tightness of the repair, operation time <180 minutes and an Objective Structured Assessment of Technical Skills score of ≥18 of 25. Skill retention was measured using a competence cumulative sum analysis of a composite binary outcome of surgical success. Secondary outcomes were cost and fabrication time of the model. RESULTS: We made a model that can be used to simulate the neurosurgical steps of spina bifida repair, including anatomic details, port insertion, placode release and descent, undermining of skin and muscular layer, and endoscopic suturing. The model was made using reusable 3-dimensional printed molds and easily accessible materials. The 1-time startup cost was €211, and each single-use, simulated myelomeningocele lesion cost €9.5 in materials and 50 minutes of working time. Two skilled endoscopic surgeons performed 6 simulated, 3-port fetoscopic repairs, whereas a third used a Da Vinci surgical robot. Operation times decreased by more than 30% from the first to the last trial. Six experiments per surgeon did not show an obvious Objective Structured Assessment of Technical Skills score improvement. Competence cumulative sum analysis confirmed competency for each surgeon. CONCLUSION: This high-fidelity, low-cost spina bifida model allows simulated dissection and closure of a myelomeningocele lesion. VIDEO ABSTRACT.

Exteriorization of the uterus reduces fetoscopic cannula-induced stress and strain: A finite element model analysis.

OBJECTIVE: To estimate stresses and strains in the uterine wall and fetal membranes with single/multi-port fetoscopy, simulating either a percutaneous access or via exteriorized uterus. STUDY DESIGN: Finite element models based on anatomical dimensions, material properties and boundary conditions were created to simulate stresses, strains and displacements on the uterine wall and fetal membranes during simulated fetal surgery either via exteriorized uterus or percutaneous approach, and with one or three cannulas. Clinically, we measured the anatomical layer thickness and cannula entry point displacement in patients undergoing single port percutaneous fetoscopy. RESULTS: Simulations demonstrate that single port percutaneous fetoscopy increases stress on the fetal membranes (+105%, 128 to 262 kPa) and uterine wall (+115%, 0.89 to 1.9 kPa) compared to exteriorized uterine access. Using three ports increases stress by 110% (148 to 312 kPa) on membranes and 113% (1.08 to 2.3 kPa) on uterine wall. Finite Element Method showed 0.75 cm uterine entry point displacement from the cutaneous entry, while clinical measurements demonstrated displacement of more than double (1.69 ± 0.58 cm), suggesting modeled measurements may be underestimations. CONCLUSION: The stresses and strains on the fetal membranes and uterus are double as high when entering percutaneously than via an exteriorized uterus. Based on what can be clinically measured, this may be an underestimation.

Ethnicity-specific BRCA1, BRCA2, PALB2, and ATM pathogenic alleles in breast and ovarian cancer patients from the North Caucasus.

BACKGROUND: Mountain areas of the North Caucasus host several large ethnic communities that have preserved their national identity over the centuries. METHODS: This study involved high-grade serous ovarian cancer (HGSOC) and breast cancer (BC) patients from Dagestan (HGSOC: 37; BC: 198), Kabardino-Balkaria (HGSOC: 68; BC: 155), North Ossetia (HGSOC: 51; BC: 104), Chechnya (HGSOC: 68; BC: 79), Ingushetia (HGSOC: 19; BC: 103), Karachay-Cherkessia (HGSOC: 13; BC: 47), and several Armenian settlements (HGSOC: 16; BC: 101). The group of BC patients was enriched by young-onset and/or family history-positive and/or bilateral and/or receptor triple-negative cases. The entire coding region of BRCA1, BRCA2, PALB2, and ATM genes was analyzed by next-generation sequencing. RESULTS: A significant contribution of BRCA1/2 pathogenic variants (PVs) to HGSOC and BC development was observed across all North Caucasus regions (HGSOC: 19-39%; BC: 6-13%). Founder alleles were identified in all ethnic groups studied, e.g., BRCA1 c.3629_3630delAG in Chechens, BRCA2 c.6341delC in North Ossetians, BRCA2 c.5351dupA in Ingush, and BRCA1 c.2907_2910delTAAA in Karachays. Some BRCA1/2 alleles, particularly BRCA2 c.9895C > T, were shared by several nationalities. ATM PVs were detected in 14 patients, with c.1673delG and c.8876_8879delACTG alleles occurring twice each. PALB2 heterozygosity was observed in 5 subjects, with one variant seen in 2 unrelated women. CONCLUSION: This study adds to the evidence for the global-wide contribution of BRCA1/2 genes to HGSOC and BC morbidity, although the spectrum of their PVs is a subject of ethnicity-specific variations. The data on founder BRCA1/2 alleles may be considered when adjusting the BRCA1/2 testing procedure to the ethnic origin of patients.

To Study the Correlation of Clinical Severity and Cytokine Storm in COVID-19 Pulmonary Embolism Patients by Using Computed Tomography Pulmonary Angiography (CTPA) Qanadli Clot Burden Scoring System.

BACKGROUND: Pulmonary embolism (PE) is a fatal form of venous thromboembolism (VTE), with an overall untreated mortality of up to 30%. Greater than 50% of patients with lower extremity proximal DVT have concurrent PE at presentation. VTE has been seen in up to one-third of patients with COVID-19 infections requiring intensive care unit (ICU) admission. The objective of this study is to determine the correlation between CT pulmonary angiography, pulmonary embolism clot burden, and the Qanadli scoring system with clinically severe COVID-19 pneumonia and cytokine storm. MATERIAL AND METHOD: 153 COVID-19 hospitalized patients who underwent CT pulmonary angiography (CTPA) for likely PE on pretest probability modified Wells criteria were enrolled. COVID-19 pneumonia was classified as URTI (upper respiratory tract infection), mild, severe, and critical COVID pneumonia. For data analysis, we categorized into two groups: (1) the non-severe group included URTI and mild pneumonia, and (2) the severe group included severe and critical pneumonia. We used the Qanadli scoring system to assess the PE percentages of pulmonary vascular obstruction using CTPA.  Results: 41.8% (64) of COVID-19 patients were diagnosed with pulmonary embolism (PE) on CTPA. The majority of 51.6% of pulmonary vascular occlusions using the Qanadli scoring system for pulmonary embolism were at segmental arterial levels. Out of 104 COVID-19 cytokine storm patients, 45 (43%) were associated with pulmonary embolism. Overall, a 25% (16) mortality rate was observed in COVID-19 patients with pulmonary embolism. DISCUSSION: The pathogenesis of hypercoagulability in COVID-19 may include direct endothelial cell invasion by the virus, microvascular inflammation, endothelial exocytosis, and endotheliitis. A meta-analysis of 71 studies to investigate the occurrence of PE on CTPA in COVID-19 patients found 48.6% in ICU settings and 65.3% of patients have clots in the peripheral pulmonary vasculature. CONCLUSIONS: There is a significant correlation between pulmonary embolism and high clot burden Qanadli CTPA scores, as well as between the severity of COVID-19 pneumonia and mortality. The association between critically ill COVID-19 pneumonia and pulmonary embolism may result in higher mortality and a poor prognostic marker.

Estimating the prevalence of pediatric hematological malignancies in Al-Madinah Al-Munawwarah, Saudi Arabia.

OBJECTIVES: To provide an updated estimate to the prevalence of pediatric hematological malignancies (HMs) in the Al-Madinah Al-Munawwara, Saudi Arabia. METHODS: This is a retrospective study that was carried out between 2016 and 2022. The study population was comprised of 171 children under 16 who had been diagnosed with HMs. The data was compiled from King Salman Medical City's Maternity and Children's Hospital, Al-Madinah Al-Munawwarah, Saudi Arabia. RESULTS: Among the 171 HM patients (64% males and 36% females), 13 subtypes were identified, with B-cell acute lymphoblastic leukemia having the highest incidence (70.3%). Acute myelomonocytic leukemia (8.7%), T-cell acute lymphoblastic leukemia (4.7%), and acute promyelocytic leukemia (3.5%) were the next most common types of HMs. Other rare cases were also found. CONCLUSION: Prevalence rate can be utilized to monitor the progression of disease incidence. Here, HMs demonstrated a pattern of increasing incidence in males over a 7-year period, with a higher rate in early childhood. There were 13 types of HMs diagnosed, with B-acute lymphocytic leukemia having the highest incidence. Although juvenile cancer is rare, it is nonetheless a significant cause of mortality in children. A successful prognosis requires prompt and accurate diagnosis and treatment.

Toxoplasma gondii, HBV, and HCV co-infection and their correlation with CD4 cells among Iranian HIV-positive patients.

INTRODUCTION: Human immunodeficiency virus (HIV/AIDS) infected patients have a higher risk of opportunistic infections (OIs) depending on their immunological status, especially CD4 + cell count. Toxoplasma gondii, hepatitis C virus (HCV), and hepatitis B virus (HBV) are important OIs among Human Immunodeficiency Virus (HIV)/Acquired Immune Deficiency Syndrome (AIDS) patients. However, little is known about co-infection of these pathogens among HIV-infected individuals and their correlation with the patient's CD4 + cell count. Hence, this study aimed to investigate the serological and molecular status of T. gondii infection among HIV-infected individuals who had co-infection with HBV and HCV infections. METHODS: A total of 100 HIV/AIDS patients in two cities in the southwest of Iran was tested for T. gondii Immunoglobulin G (IgG) and Immunoglobulin M (IgM) antibodies as well as DNA detection by polymerase chain reaction (PCR) targeting the RE gene. HBV and HCV were detected by hepatitis B surface antigen (HBsAg) test, hepatitis C antibody (HCV Ab) test, and Real-Time PCR. The number of CD4 + cell counts was determined by Flow cytometry. RESULTS: Anti-T. gondii IgG was positive in 22% of the patients, but anti-T. gondii IgM and PCR were negative in all samples. HBV and HCV were positive in 8% and 33% of the patients, respectively. Co-infections were as followed: HIV + HCV (16%), HIV + HCV + T. gondii (11%), HIV + T. gondii (5%), HIV + HBV (1%), HIV + HBV + T. gondii (1%), HIV + HBV + HCV (1%), and HIV + HBV + HCV + T. gondii (5%). A significant decline in CD4 + cell counts was found in such co-infection groups (HIV + T. gondii, HIV + HCV + T. gondii, and HIV + HBV + HCV + T. gondii) compared with the HIV mono-infection group. CONCLUSIONS: Our study showed that co-infections of T. gondii, HCV, and HBV were common among HIV-infected patients and co-infections had a negative correlation with CD4 + cell counts of the patients.

Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth.

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1-3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.

Genome-wide superior alleles, haplotypes and candidate genes associated with tolerance on sodic-dispersive soils in wheat (Triticum aestivum L.).

KEY MESSAGE: The pleiotropic SNPs/haplotypes, overlapping genes (metal ion binding, photosynthesis), and homozygous/biallelic SNPs and transcription factors (HTH myb-type and BHLH) hold great potential for improving wheat yield potential on sodic-dispersive soils. Sodic-dispersive soils have multiple subsoil constraints including poor soil structure, alkaline pH and subsoil toxic elemental ion concentration, affecting growth and development in wheat. Tolerance is required at all developmental stages to enhance wheat yield potential on such soils. An in-depth investigation of genome-wide associations was conducted using a field phenotypic data of 206 diverse Focused Identification of Germplasm Strategy (FIGS) wheat lines for two consecutive years from different sodic and non-sodic plots and the exome targeted genotyping by sequencing (tGBS) assay. A total of 39 quantitative trait SNPs (QTSs), including 18 haplotypes were identified on chromosome 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 5A, 5D, 6B, 7A, 7B, 7D for yield and yield-components tolerance. Among these, three QTSs had common associations for multiple traits, indicating pleiotropism and four QTSs had close associations for multiple traits, within 32.38 Mb. The overlapping metal ion binding (Mn, Ca, Zn and Al) and photosynthesis genes and transcription factors (PHD-, Dof-, HTH myb-, BHLH-, PDZ_6-domain) identified are known to be highly regulated during germination, maximum stem elongation, anthesis, and grain development stages. The homozygous/biallelic SNPs having allele frequency above 30% were identified for yield and crop establishment/plants m-2. These SNPs correspond to HTH myb-type and BHLH transcription factors, brassinosteroid signalling pathway, kinase activity, ATP and chitin binding activity. These resources are valuable in haplotype-based breeding and genome editing to improve yield potential on sodic-dispersive soils.

Growth factors in relation to obesity, food habits, and microbiota among healthy Saudis: preliminary results.

OBJECTIVE: Obesity is a serious problem among Saudis because of the country's affluent lifestyle. Obesity is associated with various metabolic disorders and characterized by low-grade inflammation that leads to the release of pro-inflammatory cytokines, human growth factors (GFs), lipids, aberrant adipokines, and other chemokines from adipose tissue. The objective of this study is to delineate the effects of GFs on microbiota and their relationship to body mass index (BMI) and food habits. SUBJECTS AND METHODS: In a cross-sectional study, 32 randomly selected participants (16 males and 16 females) were enrolled in a survey covering their sociodemographic information, medical history, lifestyle, and dietary practices. The information on diet, health condition, food and drink intake habits were examined under four distinct BMI categories: normal, underweight, overweight, and obese. The participants' serum samples were analyzed for the various GFs using a human magnetic 30-plex panel multiplex assay. Bioinformatics analysis was performed to investigate which bacterial taxa are enriched and to predict the functional profiles of the samples. RESULTS: Correlational studies revealed sex-based differences between GFs and microbiota. Females exhibited a significant correlation between epidermal GF (EGF) and Proteobacteria, whereas males showed a significant correlation between fibroblast GF-basic and Actinobacteria. Interestingly, a combined analysis of both sexes showed a significant correlation between EGF and vascular endothelial GF with Firmicutes. The data in the underweight group revealed a correlation between granulocyte colony-stimulating factor (G-CSF) and hepatocyte GF with Firmicutes. In the obese group, a correlation was found between G-CSF and Actinobacteria. CONCLUSIONS: Our results identified links between GFs, microbiota, and BMI in a Saudi cohort. The insights from this preliminary study will contribute to the predictive diagnosis of obesity. However, further research involving a larger cohort will be necessary to understand the mechanistic aspects of these GFs to provide biomarkers of potential obesity.

Catalytic atroposelective dynamic kinetic resolutions and kinetic resolutions towards 3-arylquinolines via SNAr.

Herein we report the catalytic atroposelective syntheses of pharmaceutically relevant 3-arylquinolines via the nucleophilic aromatic substitution (SNAr) of thiophenols into 3-aryl-2-fluoroquinolines mediated by catalytic amounts of Cinchona alkaloid-derived ureas. These reactions displayed a spectrum of dynamic kinetic resolution (DKR) and kinetic resolution (KR) characters depending upon the stereochemical stability of the starting material. Low barrier substrates proceeded via DKR while higher barrier substrates proceeded via KR. On the other hand, substrates with intermediate stabilities displayed hallmarks of both DKR and KR. Finally, we also show that we can functionalize the atropisomerically enriched quinolines into pharmaceutically privileged scaffolds with minimal observed racemization.

Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies & thalassemic syndromes.

BACKGROUND: : HPLC is one of the most important tools for accurate diagnosis of hemoglobinopathies and thalassemias. The advantage of the HPLC system is the excellent resolution, reproducibility &quantification of several normal and abnormal hemoglobin. RESULTS: BIO RAD Variant II analyzer was used. Sickle cell syndromes including double heterozygous states accounted for 56.13% of total cases. HbSS, HbS/ß0-th, HbS/ß+-th ß-thal trait comprises 29%, 6.5%, 5.1%& 10% of total cases respectively with mean MCV (fl) = 84, 68,71,64 respectively. The Mean HbA2 for ß-thal trait, HbE trait &HbE-ß thal showed 5.1 ± 1.1, 19 ± 9 & 24 ± 8 respectively. HbF is increased in 8.6% case (excluding SC syndromes & ß-thal disorders), of these 5.5% were infants & 12 cases of Aplastic Anemias. Peak P2 >7% (2.4% cases) was seen in uncontrolled diabetes mellitus which on quantification showed HbA1C = 8 ± 2.1 mmol/L. DISCUSSION: : HPLC in correlation with CBC parameters & family studies can aid in the diagnosis of majority of Hemoglobinopathies and thalassemic syndrome. The CBC & HPLC parameters of the present study are in good correlation with the research conducted by Tejinder Sing, RiouJ & Alla Joutovsky. Present study showed HPLC comprehensively characterizing HbS, A, A2, F, S, C, D from each other & was also applicable for the quantification of HbA1c for the monitoring of Diabetes Mellitus. CONCLUSION: : The merits of HPLC are small quantity of sample required, economical, less TAT, accurate categorization of HbS, HbA2 & F. But one has to be aware of the limitations and problems associated with this method due to variant hemoglobin within the same retention windows. The present findings show HPLC as an excellent & powerful diagnostic tool for the direct identification of hemoglobin variants with a high degree of precision in the quantification of normal and abnormal hemoglobin fractions.

Estimates of resource transfer via winged adult insects from the hyporheic zone in a gravel-bed river.

Hyporheic zone (HZ) locates below the riverbed providing habitat for macroinvertebrates from where the winged adult insects (i.e., hyporheic insects, HIs) emerge and bring out aquatic resources to the riparian zone. This study estimated mean daily flux as dry biomass (BM), carbon (C), and nitrogen (N) deriving from the dominant HI species Alloperla ishikariana (Plecoptera, Chloroperlidae) for a 4th-order gravel-bed river during the early-summer to summer periods. We hypothesized that HIs were an important contributor in total aquatic resources to the riparian zone. In 2017 and 2018, we set parallelly (May to August) and perpendicularly (June to October) oriented Malaise traps to catch the lateral and longitudinal directional dispersing winged adults of A. ishikariana, and other Ephemeroptera, Plecoptera, Trichoptera, and Diptera from the river and estimated the directional fluxes of them. We further split the directional fluxes as moving away or back to the channel (for lateral) and from down- to upstream or up- to downstream (for longitudinal). Alloperla ishikariana was similar to other Plecoptera species and differed clearly from Ephemeroptera and Trichoptera in directional characteristics of resources flux, suggesting that the extent and directions of HZ-derived resource transfer depend on taxon-specific flight behaviors of HIs. Contributions of A. ishikariana to the riparian zone in total aquatic C and N transfer seasonally varied and were lower in May (5%-6%) and August (2%-4%) and the highest in July (52%-70%). These conservative estimates largely increased (9% in May) after the supplementary inclusion of Diptera (Chironomidae and Tipulidae), part of which were considered HIs. We demonstrated that HZ could seasonally contribute a significant portion of aquatic resources to the riparian zone and highlighted the potential importance of HZ in nutrient balance in the river-riparian ecosystem.

First report of pulmonary disease associated with Nicoletella semolina in a horse in New Zealand.

Case history: A 9-year-old warmblood gelding with a history of chronic intermittent tachypnoea and dyspnoea was presented for evaluation and removal of a mass on the left side of the neck. A fibrous mass adherent to the left jugular vein developed and was removed surgically 6 weeks later, at which time the owner requested an evaluation of the cause of the persistent respiratory signs first noted on primary admission. Clinical findings and treatment: Clinical findings included coarse lung sounds on thoracic auscultation, tracheal wheeze, and an abnormal trans-tracheal aspirate. These findings, in addition to the results of ultrasonographic imaging of the thorax and transtracheal cytology, were suggestive of bacterial bronchopneumonia. Initial antimicrobial therapy included I/M 22 mg/kg procaine penicillin every 12 hours and I/V 6.6 mg/kg gentamicin sulphate every 24 hours. The horse's clinical signs improved within 36 hours. It was discharged after 6 days, and at the owner's request antimicrobial therapy was changed to 25 mg/kg trimethoprim/sulphadimidine to be given orally every 12 hours for 10 days. One month later, the horse had recovered and there were no further complications reported by the owner except for an occasional cough while grazing Laboratory findings: Bacterial culture of transtracheal wash fluid resulted in the isolation of Nicoletella semolina as the sole organism, later confirmed by genotyping. Attempts to subculture the organism for antimicrobial susceptibility testing were unsuccessful. Diagnosis: Infectious bronchopneumonia associated with Nicoletella semolina Clinical relevance: Further work is required to determine whether N. semolina is acting as an opportunistic commensal of the equine respiratory tract or a primary pathogen. However, this article reports the first instance in New Zealand of an association between the presence of this organism and respiratory disease in a horse.

Oxidative status and changes in the adenosine deaminase activity in experimental host infected with tropical liver fluke, Fasciola gigantica.

Fine tuning of the metabolic, physiological and immunological cues along with interplay between the biomolecules of the host and the parasite could be responsible for the successful establishment of parasitic infections. The present investigation was aimed at evaluating the oxidative status and the level of adenosine deaminase (ADA) in the serum and liver of rabbits experimentally infected with Fasciola gigantica. A significant increase in level of ROS, MDA and 4-HNE along with a decline in the SOD, CAT, GR and GST activity was evident in rabbits experimentally infected with Fasciola gigantica. However, there was an increase in the GPX activity in the sera of infected rabbits. The increased GPX activity and decreased GR activity would have resulted in the depletion of GSH, a key non-enzymatic antioxidant, in the infected animals. The level of GSSG was also found to be higher in the sera and liver tissues of the infected rabbits along with a decline in the GSH/GSSG ratio, indicating a high level of oxidative stress in the infected animals, which also showed a significant increase in the activity of the marker enzymes of liver pathology, AST and ALT. Further, a significant inhibition of the adenosine deaminase (ADA) activity in the infected rabbits was accompanied with the reduction in the level of pro-inflammatory cytokine, IL-6 while the anti-inflammatory cytokine, IL-4 level was significantly elevated. In conclusion, the F. gigantica induced significant oxidative stress as evident from the increased levels of ROS and lipid peroxidation along with the disruption of antioxidant and detoxification cascade ultimately lead to pathogenic and inflammatory responses in the experimental host. Whereas, the altered ADA activity could modulate the host's immune responses toward Th-2 type and would facilitate the successful establishment of flukes within their host, thus indicating that ADA could be exploited as a target for the development of novel anthelmintic drugs against fasciolosis.

Outbreak investigation of Candida auris at a tertiary care hospital in Karachi, Pakistan.

BACKGROUND: From September 2014, a tertiary care hospital in Karachi, Pakistan, started diagnosing 3-5 cases/month of a yeast locally identified as Saccharomyces spp. resistant to fluconazole. US Centers for Disease Control and Prevention identified the isolates as Candida auris. The Pakistan Field Epidemiology and Laboratory Training Program (FELTP) and the hospital investigated the outbreak from April 2015 to January 2016. OBJECTIVE: The aim of the outbreak investigation was to determine the risk factors and to inform measures to limit the spread of the organism in the hospital. METHODS: Medical records, nursing schedules and infection control practices were reviewed. Sixty-two age- and sex-matched hospital controls from the same wards were identified. RESULTS: Thirty cases (17 males) were identified (mean age = 51.6 years, age range = 2-91 years), case fatality was 53%. Multivariate logistic regression showed that a history of surgery within 90 days of diagnosis, admission to the emergency department and history of chronic kidney disease were significantly associated with C. auris infection. DISCUSSION: This is the report of the outbreak investigation that triggered a global exploration of C. auris as a newly identified multidrug-resistant nosocomial organism, spreading within the hospital, especially among patients with invasive procedures. Unfortunately, we could not identify any specific source of the outbreak nor stop the transmission of the organism.

Catalyst-Controlled Regioselective Chlorination of Phenols and Anilines through a Lewis Basic Selenoether Catalyst.

We report a highly efficient ortho-selective electrophilic chlorination of phenols utilizing a Lewis basic selenoether catalyst. The selenoether catalyst resulted in comparable selectivities to our previously reported bis-thiourea ortho-selective catalyst, with a catalyst loading as low as 1%. The new catalytic system also allowed us to extend this chemistry to obtain excellent ortho-selectivities for unprotected anilines. The selectivities of this reaction are up to >20:1 ortho/para, while the innate selectivities for phenols and anilines are approximately 1:4 ortho/para. A series of preliminary studies revealed that the substrates require a hydrogen-bonding moiety for selectivity.