RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. RESULTS: We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5-7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3- to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited  ~ 3.4-fold slower reaction rate (0.37 min-1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation. CONCLUSIONS: Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

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.

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.

Correlation of C-reactive protein levels, gene polymorphism and platelets count in Dengue infection.

OBJECTIVE: To determine the correlation of polymorphism in C-reactive protein gene with variation in serum levels in dengue patients. METHODS: The cross-sectional study was conducted at Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan, from October 2017 to October 2018, and comprised blood samples from dengue patients which were used to measure the serum levels of C-reactive protein. Deoxyribonucleic acid extraction followed by tetra amplification-refractory mutation system polymerase chain reaction was used to analyse the genotype variation T>G for single nucleotide polymorphism rs199953854 using allele-specific primers. Correlation of serum C-reactive protein levels with the C-reactive protein polymorphism in dengue patients was explored. Data was analysed using SPSS 21. RESULTS: Of the 200 patients, 108(54%) had very high C-reactive protein levels, 48(24%) had levels slightly higher than the upper limit, 14(7%) had low and 30(15%) had normal levels. Also, 162(81%) patients had low platelets count. Amplification of only T alleles was noted. CONCLUSIONS: C-reactive protein levels were found to be increased with suppressed platelets count in dengue patients. Single nucleotide polymorphism rs199953854 appeared to have no polymorphism.

Exploring genetic diversity and population structure of Punjab goat breeds using Illumina 50 K SNP bead chip.

Pakistan has 35 goat breeds. Moreover, the province of Punjab has highest goat population constituting 37% of country's total population with seven goat breeds including Beetal, Daira Deen Panah, Nachi, Barbari, Teddi, Pahari, and Pothwari. The diversity study of breeds warrants the documentation of breeds particularly using genome wide panel of markers, i.e., SNP chip. The objective of the current study was to fill this gap of information. Therefore, in current study we collected total of 879 unrelated goat blood samples along with data on body weight measurements; genomic DNA was extracted, and genotyping was carried out using 50 K SNP bead chip. Quality control measures were performed in Plink 1.07. Genetic diversity was observed among studied populations using heterozygosity and pairwise FST estimates, principal component analysis, admixture analysis in Plink software with visualization in Clumpak, and constructing phylogenetic tree in Mega 7 software. Moderate to high level of heterozygosity was observed among the studied populations. Coefficient of inbreeding varied from 0.0186 ± 0.0327 in Pahari to 0.183 ± 0.0715 in Barbari. Barbari and Daira Deen Panah had quite higher level of inbreeding coefficient as compared to all other breeds with value of 0.183 ± 0.0715 and 0.1378 ± 0.0741, respectively. PCA identified three steps of subdividing the seven goat breeds at various levels of K. All the seven breeds made independent clusters at various levels of PCA. Admixture analysis revealed the distinctness of Teddi and Barbari breeds. Genetic sub-structuring was observed in the admixture patterns of Beetal breed. Moreover, high level of genetic admixture was observed in Nachi, Pahari, Pothwari, and Daira Deen Panah breeds. Admixture results were further interpreted by calculating pairwise FST values. Our results provided first insights about genetic diversity of Pakistani goat breeds based on genomic data. To conclude, the enriched goat breed diversity in Pakistan could provide valuable genetic reservoir for national breeding schemes.

Knowledge, attitude and practices (KAP) of the families of b-thalassaemia children in thalassaemia centers of Rawalpindi and Islamabad, Pakistan.

The present study was designed to assess the Knowledge, Attitude and Practices (KAP) of the parents of b-thalassaemia children (410) selected from public (73.2%) and private (26.8%) thalassaemia centers of Rawalpindi-Islamabad. Qualitative and quantitative approaches were used to collect the data, which was analyzed by using SPSS. Majority of the respondents (70%) were rural young parents with no knowledge of thalassaemia before marriage. However, now 81.2% were aware about this. Majority of the respondents (89%) had the knowledge about premarital screening, 86.1% were opposed to intermarriages of carrier and 57% believed that if carrier got married then prenatal diagnosis or Chorionic villus sampling test is necessary. About 76.8% of the couples were screened and 42.2% had an experience of Chorionic villus sampling among which 20% abortions were reported. Overall 82% parents had received genetic counselling. The present study suggests that parent's regular visits and genetic counseling at thalassaemia centers have played important role about awareness.

Hypoglycemic and hypocholesterolemic activity of leave of few medicinal plants against steptozotocin induced hyperglycemia.

Diabetes mellitus (DM) is universal of the hormonal problem and Type II diabetes is foremost obstacle. Accessible management in medicine has numerous contrary paraphernalia. Medical flora shows an essential part in managing diabetes specifically in unindustrialized nations. The present study was done on leaves of Rhazya stricta Decane, Adhatoda zeylanica, Berberis lycium Royle and Olea furrrignea, whose methanolic extracts were used to check their hypoglycemic and hypolipidemic activity by using glucometer and kit method respectively in blood of male and female albino mice Balb C. Results showed that leaves of R. stricta were best for hypoglycemia (125.34±63.79mg/dl, 107.34±18.00mg/dl, 146.00±40.36mg/dl and 178.34±17.03mg/dl), hypocholesterolemia (147.88±21.83mg/dl and 125.89±14.03mg/dl) and triglycerides (103±8.88mg/dl and 89.±43.4mg/dl) in random and fasting conditions, in male and female mice respectively. All plant extract were most effective for hypoglycemia and hypocholesterolemia in female mice as compared to male mice. Moreover statistical analysis revealed that leaves of other plants were also effective but less than leaves of R. stricta. So this plant part and a combination of presently used plants can be used for further studies particularly among females by the purification of active compounds against hyperglycemia and hypercholesterolemia.

Antioxidant and hypoglycemic potential of phytogenic cerium oxide nanoparticles.

Plants provide humans with more than just food and shelter; they are also a major source of medications. The purpose of this research was to investigate the antioxidant and hypoglycemic potential of green synthesized CeONPs using Mentha royleana leaves extract. The morphological and physicochemical features of CeONPs were evaluated by UV-Visible spectrophotometry, Scanning Electron Microscopy, Energy Dispersive X-rays and Fourier-transform infrared spectrometry, Dynamic light scattering, Atomic Force Microscopy, Zeta Potential. The average size range of synthesized CeONPs diameter between 46 and 56 nm, crystalline in shape, with Polydispersity index value of 0.2 and subatomic particles mean diameter was 4.5-9.1 nm. The antioxidant capability of CeONPs was assessed using DPPH, ABTS+, hydrogen peroxide, hydroxyl radical scavenging, and reducing power tests. The hypoglycemic potential of CeONPs was investigated using alpha-amylase, alpha-glucosidase, glucose absorption by yeast cells, and antisucrase. The effective concentrations were 500 and 1000 µg/ml found good in suppressing radical species. To explore the hypoglycemic potential of CeONPs, alpha-amylase, alpha-glucosidase, glucose absorption by yeast cell, and antisucrase assays were performed. Glucose absorb by yeast cells assay was tested for three distinct glucose concentrations: 5 mmol/L, 10 mmol/L, and 25 mmol/L. Green synthesize CeONPs showed a dose-dependent response, higher concentrations of CeONPs imposed a stronger inhibitory impact on the catalytic site of enzymes. This study suggest that CeONPs could possibly binds to the charge carrying species and act as competitive inhibitor which slow down the enzyme substrate reaction and prevents enzymatic degradation. The study's findings were outstanding, which bodes well for future medicinal applications of CeONPs.

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.

Phanerochaete chrysosporium IBL-03 secretes high titers of manganese peroxidase during decolorization of Drimarine Blue K2RL textile dye.

A novel indigenous strain, Phanerochaete chrysosporium IBL-03, with high manganese peroxidase (MnP) activities was used for decolorization of a reactive textile dye, Drimarine Blue K2R, which is used extensively in textile units of Pakistan. The initial experiment was run for seven days with 0.01% (w/v) dye solution prepared in Kirk's basal nutrient medium. Samples were removed after every 24 h and the extent of dye decolorization was determined at lambda(max) of the dye. The study revealed that P. chrysosporium caused 65% decolorization of Drimarine Blue K2RL in seven days. By process optimization, 97% colour removal could be achieved in three days using 0.005% (w/v) Drimarine Blue K2RL solution at pH 4.0 and 30 degrees C in defined Kirk's medium with 0.9% (w/v) molasses and 0.2% (w/v) ammonium dihydrogen phosphate added as carbon and nitrogen sources, respectively. Manganese peroxidase was found to be the major enzyme (560 IU/mL) involved in dye decolorization of Drimarine Blue K2RL by P. chrysosporium. The dye adsorption studies showed that the dye initially adsorbed on fungal mats disappeared later on, possibly by the action of MnP secreted by the fungus in secondary metabolism.

Field Evolved Resistance in Earias vittella (Lepidoptera: Noctuidae) From Punjab, Pakistan Against Commercial Formulations of Bacillus thuringiensis kurstaki.

Transgenic commercial cotton expressing Bacillus thuringiensis (Bt) Cry endotoxins or vegetative Vip toxins provide protection to cotton against bollworm attack. Continuous exposure of these targeted pests to cry toxins and to Bt commercial spray formulations has resulted in the development of resistance through natural selection. Spotted bollworm Earias vittella (Noctuidae: Lepidoptera) is considered to be one of the most destructive pests of cotton and okra crops in South Asia including Pakistan and has developed resistance to various synthetic insecticides. In the present study, the level of resistance in field populations of the spotted bollworm E. vittella against Bt Cry toxins has been evaluated for the first time. We collected twelve populations of E. vittella from three districts of Punjab, Pakistan for testing against four commercial Bt formulations containing different strains of B. thuringiensis subspecies kurstaki (Btk) with a range of Cry toxins. Low to high levels of resistance were found in the field populations compared with a laboratory-reared susceptible population of E. vittella (resistance ratios 6 to 111-fold). These results suggest that E. vittella has developed resistance against different Cry toxins after continuous exposure to Bt cotton in field. In order to prevent field control failures, regular insecticide resistance monitoring programs are required together with the use of integrated management approaches, including the use of Bt cotton varieties expressing two or more toxins to delay the development of resistance against Bt toxins in E. vittella.

Preclinical Study on the Hepatoprotective Effect of Pollen Extract of Pinus brutia Ten. (Red Pine) in Mice and Phenolic Acid Analysis

Objectives: Many agents, including those from herbal sources, have been sought as preventives or cures for hepatotoxicity. The pollen of Pinus brutia Ten., known as red pine (Pinaceae), is used against liver diseases in Anatolian folk medicine. Materials and Methods: In the current study, pollen ethanol extract of P. brutia was investigated for its possible hepatoprotective activity using a mouse model of CCl4-induced hepatotoxicity. Swiss albino mice were divided into five groups, and extract-treated groups were compared with a silymarin-treated group as the reference. The extract was tested at 100, 200, and 300 mg/kg (b.w.). Phenolic acids were analyzed using high-performance column chromatography (HPLC) in the extracts as pollens are usually known to be rich in phenolics. Results: Our data revealed that the extract displayed the best hepatoprotection at a dose of 100 mg/kg when compared with silymarin (Legalon®), the reference drug. HPLC analysis indicated presence of protocatechuic acid (0.176 mg/g extract), p-hydroxybenzoic acid (0.001 mg/g extract), vanillic acid (VA) (0.537 mg/g extract), syringic acid (0.050 mg/g extract), and tr-cinnamic acid (0.310 mg/g extract), while the major phenolic acid was VA. Conclusion: The outcomes of this study allow us to conclude that red pine pollen extract can serve as a promising hepatoprotective agent. Among the phenolic acids analyzed in the pollen extract, vanillic acid as the major one besides some other phenolic acids detected seems to be responsible for its remarkable hepatoprotective effect.

In Silico Validation, Fabrication and Evaluation of Nano-Liposomes of Bistorta amplexicaulis Extract for Improved Anticancer Activity Against Hepatoma Cell Line (HepG2).

BACKGROUND: Bistorta amplexicaulis of the genus Polygonum (Polygonaceae) has been reported for its antioxidant and anticancer activities. However, the low cellular uptake of the compounds in its extract limits its therapeutic application. OBJECTIVES: The present study aimed at developing a nanoliposomal carrier system for B. amplexicaulis extracts for improved cellular uptake, thus resulting in enhanced anticancer activity. METHODS: Ultra Pressure Liquid Chromatography (UPLC) was used to identify major compounds in the plant extract. Nanoliposomes (NLs) were prepared by employing a thin-film rehydration method using DPPC, PEG2000DSPE and cholesterol, followed by characterization through several parameters. In vitro screening was performed against breast cancer cell line (MCF-7) and Hepatocellular carcinoma cell line (HepG-2) using MTT-assay. Raw extract and nanoliposomes were tested on Human Umbilical Vein Endothelial Cells (HUVEC). Moreover, molecular docking was performed to validate the data obtained through wet lab. RESULTS: The UHPLC method identified gallic acid, caffeic acid, chlorogenic acid and catechin as the major compounds in the extract. The NLs with a size ranging between 140-155 nm, zeta potential -16.9 to -19.8 mV and good polydispersity index of < 0.1 were prepared, with a high encapsulation efficiency of 81%. The MTT assay showed significantly (p > 0.05) high uptake and cytotoxicity of NLs as compared to the plant extract. Moreover, reduced toxicity against HUVEC cells was observed as compared to the extract. Also, the docking of identified compounds suggested a favorable interaction with the SH2 domain of both STAT3 and STAT5. CONCLUSION: Overall, the results suggest NLs as a potential platform that could be developed for the improved intracellular delivery of plant extract, thus increasing the therapeutic outcomes.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.

Epidemiology of Transfusion Transmitted Infection among Patients with ß-Thalassaemia Major in Pakistan.

Introduction. Transfusion Transmitted Infections (TTIs) continue to be a major risk in transfusions in many parts of the world. The transfusion-dependent ß-thalassaemia patients are particularly at risk of acquiring TTIs. The current study was undertaken to estimate the prevalence of TTIs in transfusion-dependent ß-thalassaemia patients. Material and Methods. A cross-sectional study of 1253 multitransfused thalassaemia major patients was conducted in five different centres of Islamabad, Rawalpindi, and Karachi. The study subjects were screened for HIV, HCV, and HBV. The screening was performed at two centres: Department of Pathology, Shaheed Zulfiqar Ali Bhutto (SZAB) Medical University, and Blood Transfusion Services, Jinnah Postgraduate Medical Centre, from July to December 2015. The confirmatory screening was performed by Chemiluminescent Immunoassay (CLIA). Results. Out of the 1253 multiple transfused patients, 317 (25.3%) were infected with TTIs. HCV was positive in 273 cases (21.7%), HBV in 38 cases (3.0%), and HIV in 6 cases (0.5%). Conclusion. HCV was the leading TTI in multitransfused thalassaemia major patients in the study. Presence of HIV in thalassaemia patients is a recent disturbing development in Pakistan. Improved regulation of blood banks including use of internationally or nationally evaluated kits will bring down the incidence of TTIs in transfusion-dependent ß-thalassaemia patients. More stringent behavioral and serological pretransfusion screening of blood for TTIs must be implemented in blood banks.

Preparation and characterization of a green nano-support for the covalent immobilization of glucoamylase from Neurospora sitophila.

The preparation of green nano supports for the covalent immobilization of enzymes is of special interest both from the economic and environmental point of view. In this contribution, we report on the synthesis of phytochemicals coated silver nanoparticles, which were used as a novel green support for the covalent immobilization of glucoamylase isolated from Neurospora sitophila. The aqueous extract of Fagonia indica was used as a source of reducing and capping agents for the reduction of silver ions into silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV-visible spectroscopy was used to detect the characteristic surface plasmon resonance bands (426, 438nm) of the silver nanoparticles. The biosynthesized silver nanoparticles were mostly spherical in shapes with an average particle size of 30-40nm (TEM and DLS measurements). X-ray diffraction and energy dispersive X-ray studies confirmed the face centered cubic crystalline form and elemental composition of the biogenic silver nanoparticles respectively. FTIR study revealed that plant polyphenolics and protein were mainly involved in the reduction and capping of silver ions. Glucoamylase from Neurospora sitophila was covalently immobilized to these nanoparticles via EDC (1-(3-(dimethylamino) propyl) 3-ethylcarbodiimidehydrochloride) coupling reaction. The immobilized enzyme exhibited higher pH and thermal stabilities as compared to the free enzyme. The kinetic constant (KM) value for the immobilized glucoamylase was higher (0.73mg/mL) than its free counterpart (0.44mg/mL), whereas the Vmax value was slightly higher for the immobilized glucoamylase. The findings of this study conclude that the newly developed green method for the synthesis of green nano-support is simple, cost effective and could be successfully used for the immobilization of various enzymes and other macromolecules.

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.

Spectrophotometric estimation of cobalt with ninhydrin.

A violet coloured complex was developed when cobalt metal reacts with ninhydrin at pH 8.2, using sodium acetate buffer solution. Absorbance of the complex was measured at 395 nm. Various factors, such as volume of the ligand used, solution pH, stability of the complex with time and interference of other metals, which effect the complex formation have been studied in detail. Present developed method can be used for the spectrophotometric estimation of cobalt with ninhydrin complex. The method is simple, selective and cheap for the determination of cobalt in very less time.

Protein intake and stress levels in nurses and housewives of Pakistan.

Stress has many biological effects on human daily life. In the present study, dietary protein intake was correlated with the investigated stress levels of nurses and housewives of the targeted urban population. Age group ranged from 30 to 45 years and both the groups belonged to middle socioeconomic status. After calculations of environmental, psychological and physiological stresses, it was observed that the levels of stress in housewives were significantly higher than those of nurses. Recommended dietary allowances, RDA and actual protein intakes, API were also compared in both the groups. The found protein intake was less in housewives as compared to that of nurses.