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.

Antecedent infections, recent developments and future directions in Guillain-Barré syndrome.

The Guillain-Barré syndrome is an autoimmune polyradiculoneuropathy causing symmetrical weakness of limbs. After poliomyelitis, it is the second most common cause of paralysis, with an annual incidence of 0.84-1.91 per 100,000 individuals. The syndrome affects both men and women, showing a male preponderance. Campylobacter jejuni, epstein-barr virus, cytomegalovirus, mycoplasma pneumoniae and haemophilus influenzae are amongst the most common causative agents of Guillain-Barré syndrome. Several immunological and genetic factors have been recognised as the risk factors. Human leukocyte antigen, cluster of differentiation 1, and tumour necrosis factor-alpha alleles are among the frequently investigated loci in Guillain-Barré syndrome. Genome-wide association studies have found no significant association of Guillain-Barré syndrome with common variants. Many vaccines against Campylobacter jejuni infection have been proposed, but there are concerns about the efficacy and safety of these vaccines. So far, there is no approved vaccine against Campylobacter jejuni.

Incidence of malarial infection and response to antimalarial drugs at Districts Lower Dir and Swat of Khyber Pakhtunkhwa, Pakistan.

Malaria is the leading cause of mortality and morbidity all over the world. Several antimalarial drugs are available for the treatment of malaria. The main objective of this study was to investigate the incidence of malarial infection and the use of prescribed antimalarial drugs. A cross-sectional study was carried out to collect quantitative data from selected sites in District Lower Dir and Swat of Malakand Division Khyber Pakhtunkhwa (K.P.), Pakistan. Screening of selected patients was performed using both thick and thin films and was observed with the help of a light microscope. In this study, a total of 2517 blood samples were tested. Overall positive infection was 12% Plasmodium vivax (99.07%) and Plasmodium falciparum (0.92%). Our results evaluate that infection with Plasmodium vivax was higher than Plasmodium falciparum. No other Plasmodium species or mixed infections were observed. The rate of infection was more frequent in males as compared to female patients. The highest percentage was recorded in the summer season (35.07%), while the lowest was documented in the winter (11.7%). Out of 325 patients, 311 (95.7%) were treated with Chloroquine, and the remaining were treated with Artemether. Chloroquine was used as a drug of choice for Plasmodium vivax infection. The present study concludes that Plasmodium vivax and Plasmodium falciparum are the two common agents for malaria in Malakand Division. However, Plasmodium vivax was dominant over Plasmodium falciparum. The infection rate was high in males from District Lower Dir during the summer season.

Study of Guillain-Barre syndrome etiology in Pakistani patients.

OBJECTIVE: To examine clinical features, biochemical markers, demographic features, antecedent infections, frequency and treatment strategies related to Guillain-Barré syndrome. METHODS: The case-control study was conducted at the Pakistan Institute of Medical Sciences, Islamabad, Pakistan, and the District Headquarters Hospital, Rawalpindi, Pakistan, from 2018 to 2020, and comprised Guillain-Barré syndrome patients in group A and healthy controls in group B. The patients were diagnosed on the basis of clinical presentation, nerve conduction study, electromyography, cerebrospinal fluid analysis and biochemical profile. Data was analysed using SPSS 23. RESULTS: Of the 167 subjects, 90(54%) were in group A and 77(46%) were in group B. The mean age of group A was 40.20±14.90 years, while there were 61(67.7%) males and 29(32.2%) females compared to 50 (64.93%) males and 27 (35.06%) females with mean age 38.40±12.34 years in group B. Acute inflammatory demyelinating polyneuropathy was the most common electrophysiological variant of Guillain-Barré syndrome 41(46%). There was significant difference in mean interleukin-17 levels between group A 23.12±3.41 pg/ml and group B 8.82±2.49 (p<0.05). Gastrointestinal infection was the most common preceding infection 51(56.66%). The mean cerebrospinal fluid protein was 100.83±51.32g/dl and albumiocytologic dissociation was found in all the four variants (p= 0.005). CONCLUSION: Guillain-Barré syndrome affected patients regardless of age, while males were more affected than females. Majority of the patients had an antecedent infection before disease onset. Increased levels of interleukin-17 showed involvement of autoimmunity. Albuminocytologic dissociation differentiated it from poliomyelitis.

Carbamoyl Phosphate Synthase Subunit CgCPS1 Is Necessary for Virulence and to Regulate Stress Tolerance in Colletotrichum gloeosporioides.

Glomerella leaf spot (GLS) is a severe infectious disease of apple whose infective area is growing gradually and thus poses a huge economic threat to the world. Different species of Colletotrichum including Colletotrichum gloeosporioides are responsible for GLS. For efficient GLS control, it is important to understand the mechanism by which the cruciferous crops and C. gloeosporioides interact. Arginine is among one of the several types of amino acids, which plays crucial role in biochemical and physiological functions of fungi. The arginine biosynthesis pathway involved in virulence among plant pathogenic fungi is poorly understood. In this study, CgCPS1 gene encoding carbamoyl phosphate synthase involved in arginine biosynthesis has been identified and inactivated experimentally. To assess the effects of CgCPS1, we knocked out CgCPS1 in C. gloeosporioides and evaluated its effects on virulence and stress tolerance. The results showed that deletion of CgCPS1 resulted in loss of pathogenicity. The Δcgcps1 mutants showed slow growth rate, defects in appressorium formation and failed to develop lesions on apple leaves and fruits leading to loss of virulence while complementation strain (CgCPS1-C) fully restored its pathogenicity. Furthermore, mutant strains showed extreme sensitivity to high osmotic stress displaying that CgCPS1 plays a vital role in stress response. These findings suggest that CgCPS1 is major factor that mediates pathogenicity in C. gloeosporioides by encoding carbamoyl phosphate that is involved in arginine biosynthesis and conferring virulence in C. gloeosporioides.

Effect of leaf extracts of Taraxacum officinale on CCl4 induced hepatotoxicity in rats, in vivo study.

Taraxacum officinale L is a medicinal plant, which has enormous medicinal values against various types of liver disorders and it has traditionally been used for the treatment of liver problems by people from the South East Asia. Previously we have screened the crude methanolic extract of T. officinale against cytotoxicity induced by CCl4. Present study was designed to compare the protective effect of ethanolic and n-hexane extract of leaves in carbon tetrachloride (CCl4) induced liver toxicity in rats. The extract (200 mg/kg and 400mg/kg body weight) along with silymarin (100 mg/kg) a standard drug was administered to experimental animals. It was observed that ethanolic plant extract has significantly reduced the negative effect of CCl4 as compared to n-hexane extract and effect of extract was increased with increasing dose level. Although both leaf extracts decreased the concentration of TBARS, H2O2 and nitrite contents which enhance due to CCl4 toxicity but effect was higher in ethanolic extract. The results clearly indicated that Taraxacum officinale ethanolic leaves extract has better protective effect against CCl4 induced liver tissues toxicity. This claim was also supported by histopathological results obtained during this study and this might be due to presence of various polar phytochemicals that might be more prevent in this extract.

Exploring thermophilic cellulolytic enzyme production potential of Aspergillus fumigatus by the solid-state fermentation of wheat straw.

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K m, and 8.53 µM/mL/min V max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K m, and 7.29 µM/mL/min V max. In the case of ß-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on ß-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of ß-glucosidase was 55 °C, optimum pH was 4.8, K m was 4.92 mM, and V max 6.75 µM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K m and V max values indicated that endoglucanase, exoglucanase, and ß-glucosidase have good affinity for their substrates.

Presence of HCV RNA in peripheral blood mononuclear cells may predict patients response to interferon and ribavirin therapy.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) is considered a hepatotropic virus, but it can repli.cate in peripheral blood mononuclear cells (PBMCs), which influence the sustained virological response (SVR) of the patients, as well as relapse in successfully treated patients. The main objective of this study was to establish the importance of PBMC HCV RNA detection as a primary test to declare the patient as a responder, and the secondary objective was to investigate the risk of non-SVR or relapse in individuals who showed an end-of-treatment (ETR). DESIGN AND SETTINGS: Blood samples were collected after the completion of 6 months of therapy, and they were collected 6 months after the completion of treatment. PATIENTS AND METHODS: A total 103 patients infected with the 3a genotype of HCV and those who were treated with interferon-a-2b and ribavirin for 24 weeks were selected. HCV RNA in plasma of at the end of treatment and 6 months after the completion of treatment was determined with the help of quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Of the 103 patients, 74.8% (number [n]=77) were end-of-treatment responders, while 25.2% (n=26) were nonresponders. Seventy-seven responders were tested for HCV RNA in their PBMCs. The HCV RNA was detected in the PBMCs of 29 patients (37.7%). After 6 months of the end of treatment, 15 (19.5%) of 77 ETR patients showed virological relapse, while 62 (80.5%) patients attained SVR. Relapse appeared significantly more often in patients with HCV RNA in their PBMCs at the ETR stage when compared to the patients who did not have the viral RNA (34.5% versus 10.4%, respectively; R2=6.67, P=.01; odds ratio [OR]: 1.3; 95% confidence interval [CI]=1.032-1.811). CONCLUSION: Patients with HCV RNA in their PBMCs after attaining an ETR are more likely to show relapse as compared to patients who are negative for viral RNA in PBMCs at the ETR stage.

Production, purification, and characterization of exoglucanase by Aspergillus fumigatus.

Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K(m) and V(max) were 4.34 mM and 7.29 µM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.