Purification and characterization of novel isoforms of the polyphenol oxidase from Malus domestica fruit pulp.

Polyphenol oxidases (PPOs), belong to the group of oxidoreductases that are copper containing enzymes and are responsible for plant browning. PPOs are extensively distributed in plant kingdom and can oxidize wide range of aromatic compounds of industrial importance. The aim of this study was purification and characterization of PPO isoforms from the fruit pulp of Golden delicious apple. High performance liquid chromatography was used to purify the two novel isoforms of PPO and further their molecular weights (45 and 28 kDa) were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified isoforms have optimum pH (6.5), optimum temperature (40°C), the Vmax (4.45 µM/min) and Km (74.21 mM) with catechol substrate. The N-terminal microsequences of both PPO isoforms were determined using a pulse liquid protein sequencer and found to be AKITFHG (28 kDa) and APGGG (45 kDa). Polyphenol oxidases are efficiently used in the pharmaceutical, paper and pulp, textiles and food industries. Recently, the PPOs have been used for bioremediation and in the development of biosensors.

The 21st century disaster: The COVID-19 epidemiology, risk factors and control.

The 21st century will be indelible in the world as ruin of the outbreak of COVID-19 was arose in Wuhan, China has now spread all over the world, up to August 2020. This study was based on the factors affecting the epidemiology of this virus in human societies of global concern. We studied the articles published in journals on various aspects of nCoVID19. The Wikipedia and WHO situation reports have also been searched out for related information. Outcomes were followed up until 2020. The COVID-19 is a virus with pandemic potential which may continue to cause regular infection in human. The pandemic outbreak of COVID-19 threatened public health across the globe in form of system as reflected in the shape of emergency. Approximately 21 million humans are infected and 759,400 have lost their lives till 2020 in all over the world. We have described epidemiological features, reservoirs, transmission, incubation period, rate of fatality, management including recent clinical chemotherapeutic approach and preventive measurements and masses which are at risk of COVID19. This virus causes viral pneumonia when it attacks on respiratory system and multiple failure which can leads to life threatening complications. It is believed to be zoonotic importance although it is not clear from which animal and how it is transmitted. Zoonotic transmission of COVID-19 has not yet known by science. The current study will help to establish a baseline for early effective control of this rapidly spreading severe viral illness. The available data on COVID-19 indicates that older males with comorbidities would have been more infected, which can result in severe respiratory complications. Implementation of preventive measurements, investigation of proper chemotherapeutics and detection of cross species transmission agents must be ensured.

B-cell-specific MhcII regulates microbiota composition in a primarily IgA-independent manner.

Background: The expression of major histocompatibility complex class II (MhcII) molecules on B cells is required for the development of germinal centers (GCs) in lymphoid follicles; the primary sites for the generation of T-cell-dependent (TD) antibody responses. Peyer's patches (PPs) are secondary lymphoid tissues (SLOs) in the small intestine (SI) that give rise to high-affinity, TD antibodies (mainly immunoglobulin A (IgA)) generated against the microbiota. While several studies have demonstrated that MhcII antigen presentation by other immune cells coordinate TD IgA responses and regulate microbiota composition, whether or not B-cell-specific MhcII influences gut microbial ecology is unknown. Methods: Here, we developed a novel Rag1 -/- adoptive co-transfer model to answer this question. In this model, Rag1 -/- mice were reconstituted with naïve CD4+ T cells and either MhcII-sufficient or MhcII-deficient naïve B cells. Subsequent to this, resulting shifts in microbiota composition was characterized via 16S rRNA gene sequencing of SI-resident and fecal bacterial communities. Results: Results from our experiments indicate that SLO development and reconstitution of an anti-commensal TD IgA response can be induced in Rag1 -/- mice receiving T cells and MhcII-sufficient B cells, but not in mice receiving T cells and MhcII-deficient B cells. Results from our 16S experiments confirmed that adaptive immunity is a relevant host factor shaping microbial ecology in the gut, and that its impact was most pronounced on SI-resident bacterial communities. Conclusion: Our data also clearly establishes that MhcII-mediated cognate interactions between B cells and T cells regulates this effect by maintaining species richness in the gut, which is a phenotype commonly associated with good health. Finally, contrary to expectations, our experimental results indicate that IgA was not responsible for driving any of the effects on the microbiota ascribed to the loss of B cell-specific MhcII. Collectively, results from our experiments support that MhcII-mediated antigen presentation by B cells regulates microbiota composition and promotes species richness through an IgA-independent mechanism.

Computational Assessment of Microrotation and Buoyancy Effects on the Stagnation Point Flow of Carreau-Yasuda Hybrid Nanofluid with Chemical Reaction Past a Convectively Heated Riga Plate.

The present framework deliberated the mixed convection stagnation point flow of a micropolar Carreau-Yasuda hybrid nanoliquid through the influence of the Darcy-Forchheimer parameter in porous media toward a convectively heated Riga plate. In this investigation, blood is used as a base liquid and gold (Au) and copper (Cu) are the nanoparticles. The main novelty of the present investigation is to discuss the transmission of heat through the application of thermal radiation, viscous dissipation, and the heat source/sink on the flow of a micropolar Carreau-Yasuda hybrid nanoliquid. Further, the results of the chemical reaction are utilized for the computation of mass transport. Brownian motion and thermophoretic phenomena are discussed in the current investigation. The current problem is evaluated by using the connective and partial slip conditions and is formulated on the basis of the higher-order nonlinear PDEs which are converted into highly nonlinear ODEs by exploiting the similarity replacement. In the methodology section, the homotopic analysis scheme is employed on these resulting ODEs for the analytical solution. In the discussion section, the results of the different flow parameters on the velocity, microrotation, energy, and mass of the hybrid nanofluid are computed against various flow parameters in a graphical form. Some of the main conclusions related to the present investigation are that the velocity profile is lowered but the temperature is augmented for both nanoparticles volume fractions. It is notable that the skin friction coefficient is reduced due to the higher values of the Darcy-Forchheimer parameter. Further, the rising performance of the hybrid nanofluid Nusselt number is determined by the radiation parameter.

Gluten-free diet exposure prohibits pathobiont expansion and gluten sensitive enteropathy in B cell deficient JH-/- mice.

In humans, celiac disease (CeD) is a T-cell-driven gluten-sensitive enteropathy (GSE) localized to the small bowel (duodenum). The presence of antibodies specific for gluten- and self-antigens are commonly used diagnostic biomarkers of CeD and are considered to play a role in GSE pathogenesis. Previously, we have described an apparent T-cell-mediated GSE in CD19-/- mice, which develop weak and abnormal B cell responses. Here, we expand on this observation and use a mouse model of complete B cell deficiency (JH-/- mice), to show that absence of a humoral immune response also promotes development of a GSE. Furthermore, 16S analysis of microbial communities in the small intestine demonstrates that a gluten-free diet suppresses the expansion of anaerobic bacteria in the small intestine and colonization of the small intestine by a specific pathobiont. Finally, we also observe that SI enteropathy in mice fed a gluten-rich diet is positively correlated with the abundance of several microbial peptidase genes, which supports that bacterial metabolism of gluten may be an important driver of GSE in our model. Collectively, results from our experiments indicate that JH-/- mice will be a useful resource to investigators seeking to empirically delineate the contribution of humoral immunity on GSE pathogenesis, and support the hypothesis that humoral immunity promotes tolerance to gluten.

Defective humoral immunity disrupts bile acid homeostasis which promotes inflammatory disease of the small bowel.

Mucosal antibodies maintain gut homeostasis by promoting spatial segregation between host tissues and luminal microbes. Whether and how mucosal antibody responses influence gut health through modulation of microbiota composition is unclear. Here, we use a CD19-/- mouse model of antibody-deficiency to demonstrate that a relationship exists between dysbiosis, defects in bile acid homeostasis, and gluten-sensitive enteropathy of the small intestine. The gluten-sensitive small intestine enteropathy that develops in CD19-/- mice is associated with alterations to luminal bile acid composition in the SI, marked by significant reductions in the abundance of conjugated bile acids. Manipulation of bile acid availability, adoptive transfer of functional B cells, and ablation of bacterial bile salt hydrolase activity all influence the severity of small intestine enteropathy in CD19-/- mice. Collectively, results from our experiments support a model whereby mucosal humoral immune responses limit inflammatory disease of the small bowel by regulating bacterial BA metabolism.

Prevalence of drug-resistant microbes in sepsis cases of catheter and fistula based haemodialysis.

BACKGROUND: Chronic stage renal disease is a severe disease of the kidney which affects people globally. According to the global burden of diseases in 2010, this disease has caused more deaths worldwide and due to the high death rate, the ESRD (end-stage renal disease) is now ranked up from 27th to 18th range in the list. METHODOLOGY: Dialysis samples were collected from the Haripur city and surrounding areas. Samples were inoculated on different selective media for bacterial growth. In addition, different biochemical tests were also performed for identification, where as the resistance genes were identified through a polymerase chain reaction. RESULT: Out of the total 100 dialysis patient's blood samples, only 17 showed the presence of gram-positive bacteria i.e., Staphylococcus aureus while two shown the presence of gram-negative bacteria i.e., Klebsiella pneumoniaeee and Pseudomonas aeruginosa. While in molecular identification two antibiotic resistance genes muc and mecA belong to the staphylococcus strain shown their presence. CONCLUSION: A high infection rate has been observed in fistula-based hemodialysis (17(77.27%)) as compares to catheter-based hemodialysis (5(22.3%) with no significant difference of incidence between the groups (p > 0.05).

Bioherbicidal ability and weed management of allelopathic methyl esters from Lantana camara.

Allelochemicals are secondary metabolites which are not edible and can be used as growth regulators and bio-herbicides. The goal of current study was to assess allelopathic ability of Lantana camara (Sage-plant) flowers against weeds viz. Avena fatua (Wild oat), Euphorbia helioscopia (Sun-spurge), Chenopodium album (Goosefoot), Phalaris minor (Canary-grass), and Rumex dentatus (Knotweed). Bioassay analysis of three methanolic fractions of the Combiflash from L. camara was performed at 50%, 75% and 100% concentration using germination percentage parameters, inhibition of plumule and radicle size. The fraction II of Combiflash strongly suppressed all weeds with negligible effect on T. aestivum. Gas chromatography-mass spectroscopy was conducted for the fraction, and isolated compounds were used to perform bioassays. From fraction II GC-MS detected four methyl esters of allelopathic fatty acid viz. Methyl oleate, methyl palmitate, methyl stearate and methyl linoleate. The evaluation of physiological effects of the bioassay revealed substantial suppression of chlorophyll, antioxidant enzymes (superoxide, dismutase peroxidase) and protein material in all weeds by methyl palmitate. Bioassay activity and study of physiological parameters revealed that the effective bio-herbicidal compound in Lantana camara flowers is methyl palmitate. This is the first time that methyl palmitate (a fatty acid methyl ester) has been related to herbicidal activity in L. camara flowers. It is proposed that field studies based on hormesis research and the mechanism of action of this compound be carried out.

Identification of Lead Compounds against Scm (fms10) in Enterococcus faecium Using Computer Aided Drug Designing.

(1) Background: Enterococcus faecium DO is an environmental microbe, which is a mesophilic, facultative, Gram-positive, and multiple habitat microorganism. Enterococcus faecium DO is responsible for many diseases in human. The fight against infectious diseases is confronted by the development of multiple drug resistance in E. faecium. The focus of this research work is to identify a novel compound against this pathogen by using bioinformatics tools and technology. (2) Methods: We screened the proteome (accession No. PRJNA55353) information from the genome database of the National Centre for Biotechnology Information (NCBI) and suggested a potential drug target. I-TASSER was used to predict the three-dimensional structure of the protein, and the structure was optimized and minimized by different tools. PubChem and ChEBI were used to retrieve the inhibitors. Pharmacophore modeling and virtual screening were performed to identify novel compounds. Binding interactions of compounds with target protein were checked using LigPlot. pkCSM, SwissADME, and ProTox-II were used for adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. (3) Results: Novel selected compounds have improved absorption and have better ADMET properties. Based on our results, the chemically identified inhibitor ZINC48942 targeted the receptor that can inhibit the activity of infection in E. faecium. This research work will be beneficial for the scientific community and could aid in the design of a new drug against E. faecium infections. (4) Conclusions: It was observed that novel compounds are potential inhibitors with more efficacy and fewer side effects. This research work will help researchers in testing and identification of these chemicals useful against E. faecium.

Clinical and Laboratory Characteristics of Primary Immunodeficiency Patients from a Tertiary Care Center in Pakistan.

OBJECTIVE: The aim of this study was to describe and identify clinical presentation of primary immunodeficiency disorders (PIDs). Characteristic quantitative and qualitative immunological abnormalities have been described which help in establishing a definitive PID diagnosis. METHODS: This was a cross sectional study conducted in the Immunology department of the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from Jan 2016 to Dec 2018. Sixty patients of different PIDs including humoral defects, combined immunodeficiency, phagocytic defects and other miscellaneous disorders, were diagnosed over a period of 3 years in our institute. Their clinical presentation and laboratory data are presented in this study. RESULTS: In 3 years, 40 (66%) males and 20 (33%) females were diagnosed, with 13 (21.6%) patients of humoral deficiency, 22 (36.6%) of severe combined immunodeficiency, 18 (30%) of phagocytic defects and 7 (11.6%) of other miscellaneous disorders. Maximum patients belonged to Punjab province, i.e., 23 (38.3%). Their mean age for initiation of symptoms was 7±12.6 months, while diagnosis was made at mean age of 26±39.28 months, in all groups combined. Respiratory infections were commonest presentation, in 46 (76.6%) patients. Also 46 (76.6%) patients had consanguineous parents. Presence of family history of PID in 27 (45%) patients was not associated with an earlier diagnosis (p 0.955). Each group of patients carried characteristic laboratory findings. CONCLUSIONS: PIDs should be suspected in offsprings with warning signs coming from consanguineous parents. There is a need to introduce genetic diagnosis of PIDs in order to timely diagnose less characteristic PID presentations.

Gut Antibody Deficiency in a Mouse Model of CVID Results in Spontaneous Development of a Gluten-Sensitive Enteropathy.

Primary immunodeficiencies are heritable disorders of immune function. CD19 is a B cell co-receptor important for B cell development, and CD19 deficiency is a known genetic risk factor for a rare form of primary immunodeficiency known as "common variable immunodeficiency" (CVID); an antibody deficiency resulting in low levels of serum IgG and IgA. Enteropathies are commonly observed in CVID patients but the underlying reason for this is undefined. Here, we utilize CD19-/- mice as a model of CVID to test the hypothesis that antibody deficiency negatively impacts gut physiology under steady-state conditions. As anticipated, immune phenotyping experiments demonstrate that CD19-/- mice develop a severe B cell deficiency in gut-associated lymphoid tissues that result in significant reductions to antibody concentrations in the gut lumen. Antibody deficiency was associated with defective anti-commensal IgA responses and the outgrowth of anaerobic bacteria in the gut. Expansion of anaerobic bacteria coincides with the development of a chronic inflammatory condition in the gut of CD19-/- mice that results in an intestinal malabsorption characterized by defects in lipid metabolism and transport. Administration of the antibiotic metronidazole to target anaerobic members of the microbiota rescues mice from disease indicating that intestinal malabsorption is a microbiota-dependent phenomenon. Finally, intestinal malabsorption in CD19-/- mice is a gluten-sensitive enteropathy as exposure to a gluten-free diet also significantly reduces disease severity in CD19-/- mice. Collectively, these results support an effect of antibody deficiency on steady-state gut physiology that compliment emerging data from human studies linking IgA deficiency with non-infectious complications associated with CVID. They also demonstrate that CD19-/- mice are a useful model for studying the role of B cell deficiency and gut dysbiosis on gluten-sensitive enteropathies; a rapidly emerging group of diseases in humans with an unknown etiology.

Does MHC heterozygosity influence microbiota form and function?

MHC molecules are essential for the adaptive immune response, and they are the most polymorphic genetic loci in vertebrates. Extreme genetic variation at these loci is paradoxical given their central importance to host health. Classic models of MHC gene evolution center on antagonistic host-pathogen interactions to promote gene diversification and allelic diversity in host populations. However, all multicellular organisms are persistently colonized by their microbiota that perform essential metabolic functions for their host and protect from infection. Here, we provide data to support the hypothesis that MHC heterozygote advantage (a main force of selection thought to drive MHC gene evolution), may operate by enhancing fitness advantages conferred by the host's microbiome. We utilized fecal 16S rRNA gene sequences and their predicted metagenome datasets collected from multiple MHC congenic homozygote and heterozygote mouse strains to describe the influence of MHC heterozygosity on microbiome form and function. We find that in contrast to homozygosity at MHC loci, MHC heterozygosity promotes functional diversification of the microbiome, enhances microbial network connectivity, and results in enrichment for a variety of microbial functions that are positively associated with host fitness. We demonstrate that taxonomic and functional diversity of the microbiome is positively correlated in MHC heterozygote but not homozygote animals, suggesting that heterozygote microbiomes are more functionally adaptive under similar environmental conditions than homozygote microbiomes. Our data complement previous observations on the role of MHC polymorphism in sculpting microbiota composition, but also provide functional insights into how MHC heterozygosity may enhance host health by modulating microbiome form and function. We also provide evidence to support that MHC heterozygosity limits functional redundancy among commensal microbes and may enhance the metabolic versatility of their microbiome. Results from our analyses yield multiple testable predictions regarding the role of MHC heterozygosity on the microbiome that will help guide future research in the area of MHC-microbiome interactions.

Immunophenotypic characterisation of morphologically diagnosed cases of Acute Myeloid Leukaemia (AML).

OBJECTIVE: To determine immunophenotypic pattern in newly diagnosed cases of acute myeloid leukaemia by flow cytometry and its correlation with morphological findings. METHODS: This study was conducted at Haematology (Pathology) department, Army Medical College, in collaboration with Immunology Department Armed Forces Institute of Pathology, Rawalpindi from 16 November 2016 to 16 November 2017. One hundred and six patients of both genders and all age groups diagnosed as acute myeloid leukaemia were included in the study. Demographic data was noted. Complete blood counts, bone marrow examination and cytochemical stains were carried out and evaluated microscopically for blast percentage and morphology. Immunophenotyping was performed by flow cytometry using standard panel on peripheral blood or bone marrow samples. The surface and cytoplasmic antigens of interest were analysed and correlated with morphological findings. RESULTS: The most commonly expressed antigens were CD13, CD33, CD45 and HLA-DR. Almost all blasts expressed CD45 with no remarkable difference among the subtypes of AML. The mean positivity for CD13 among all AML subtypes was 57% and for CD33 was 67%. Aberrant expression of CD7 and CD19 were expressed in 26.4% and 1.1% of all cases respectively. There was concordance rate of 90% between morphology and FCM in our study. CONCLUSION: Flow cytometric analysis of acute leukaemia done by a combination of patterns and intensity of antigen expression improves diagnostic yield in AML. CD13, CD33 and CD45 are the most frequently expressed antigens in AML. Our findings suggest a 90% concordance between morphology and flow cytometry. It is pertinent to conclude that flow cytometry results interpreted with morphology are complementary.

Evaluation of Serum Free Light Chain in Diagnosis and Monitoring of Plasma Cell Disorders.

OBJECTIVE: To determine diagnostic accuracy of serum free light chain assay compared to serum and urine protein electrophoresis in plasma cell disorders. STUDY DESIGN: Descriptive cross-sectional study. PLACE AND DURATION OF STUDY: This study was conducted in the Immunology Department, Armed Forces institute of Pathology (AFIP), Rawalpindi, from May 2017 to May 2018. METHODOLOGY: Patients referred to AFIP for diagnosis of plasma cell disorders or for monitoring while receiving treatment were included in study. They were tested for serum protein electrophoresis (SPE), urine protein electrophoresis (UPE), immunofixation (IF), and serum free light chain assay (sFLC). IF was used as the reference standard. Test results were compared in terms of sensitivity, specificity, positive or negative predictive value, and accuracy index. RESULTS: During the study period 220 patients were tested for plasma cell disorders. One hundred and sixty-seven patients tested positive. One hundred twenty-nine patients had multiple myeloma, 13 plasmacytoma, 11 monoclonal gammopathy of undetermined significance, 6 amyloidosis, 6 POEMS, and 2 Waldenstrom macroglobulinemia. SPE had a sensitivity of 70.5%, specificity of 100%; sFLC had a sensitivity of 87%, specificity of 81%; and UPE had a sensitivity of 23.5%, specificity of 97%. Accuracy index was 80.5% for SPE, 85% for sFLC, and 54% for UPE. When taken together, SPE and UPE had a combined sensitivity of 72%, specificity 97%, and accuracy index 80.5%. SPE and sFLC had combined sensitivity of 98.6%, specificity 84.3%, and accuracy index 94%. CONCLUSION: Combination of SPE and sFLC had the highest sensitivity and accuracy index for diagnosis and monitoring of plasma cell disorders compared with conventional tests.

Significance of Testing Anti-Thyroid Autoantibodies in Patients with Deranged Thyroid Profile.

BACKGROUND: We hypothesized that anti-thyroid antibodies are more often positive in individuals with deranged thyroid profile. METHODS: This prospective cohort was done in Immunology Department, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from Jan 2017 to Oct 2017. All the samples that were referred to us for testing anti-thyroid antibodies (anti-TPO or anti-TG antibodies) and thyroid profile were included in the study. There were no exclusion criteria. Tests for anti-thyroid antibodies were performed by ELISA and thyroid profile by chemiluminescence. SPSS 23.0 was used for statistical analysis. RESULTS: Over a course of a ten-month study period, we received a total of 316 serum samples for anti-TPO/TG antibodies along with thyroid profile testing (TSH). These included 115 males (36.4%) and 201 females (63.6%). Their age ranged from 3 to 89 years (mean ± SD, 42.22 ± 18.09). Anti-TPO antibodies were more often positive when TSH was deranged (p value 0.001). Anti-TPO antibodies are more often raised in females, in terms of both prevalence (p 0.001) and mean rank (p 0.002). CONCLUSION: As anti-thyroid antibodies are more often present when TSH is deranged, such individuals should be screened for anti-thyroid antibodies. This importance of screening is compounded by the fact that anti-thyroid antibodies may be positive in a significant percentage of elderly people.

Protective effects of Digera muricata (L.) Mart. on testis against oxidative stress of carbon tetrachloride in rat.

Oxidative stress induced by carbon tetrachloride (2 ml/kg body weight i.p.) in rat substantially decreases the increase in body weight and relative testis weight. It also markedly increases the level of TBARS and nitrites along with corresponding decrease in reduced glutathione and various antioxidant enzymes in testis, i.e., catalase, peroxidase, superoxide dismutase and glutathione peroxidase. Serum level of testosterone, luteinizing hormone and follicle stimulating hormone was significantly decreased while estradiol and prolactin were increased with carbon tetrachloride treatment. Histopathology of CCl(4)-treated rats indicated the partial degeneration of germ and Leydig cells along with deformities in spermatogenesis. Supplementation of Digera muricata (100, 150, 200mg/kg body weight orally) once a week for 16 weeks results in decrease of TBARS and nitrite, while increase in antioxidant enzymes; catalase, peroxidase, superoxide dismutase, GSH-Px and GSH contents. Serum level of testosterone, luteinizing hormone, follicle stimulating hormone, estradiol, prolactin, histology, body weight and relative testis weight was also concomitantly restored to near normal level by D. muricata supplementation to carbon tetrachloride intoxicated rat. The results clearly demonstrate that D. muricata treatment augments the antioxidants defense mechanism against carbon tetrachloride induced toxicity and provides evidence that it may have a therapeutic role in free radical mediated diseases.

Chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an X-linked/ autosomal recessive primary immunodeficiency disorder characterized by recurrent infections. The diagnosis is primarily based on simple Nitrobluctetrazolium dye reduction test. We describe here an unusual case of an 8 year old girl, as the disease is X-linked in most of the cases.

CD5-positive acute lymphoblastic leukemia.

CD5-positive B-ALL is a rare variant of Acute Lymphoblastic Leukemia (ALL). In literature, only three cases have been reported so far. This fourth case report describes a young lady who was diagnosed as ALL (L-2) on bone marrow examination and was found to be CD5 positive B-cell acute lymphoblastic leukemia on immunophenotyping. Cytogenetic analysis revealed translocation t(9:22).