Cellulolytic and Ethanologenic Evaluation of Heterotermes indicola's Gut-Associated Bacterial Isolates.

Cellulose is the basic component of lignocellulosic biomass (LCB) making it a suitable substrate for bioethanol fermentation. Cellulolytic and ethanologenic bacteria possess cellulases that convert cellulose to glucose, which in turn yields ethanol subsequently. Heterotermes indicola is a subterranean termite that causes destructive damage by consuming wooden structures of infrastructure, LCB products, etc. Prospectively, the study envisioned the screening of cellulolytic and ethanologenic bacteria from the termite gut. Twenty six bacterial strains (H1-H26) based on varied colonial morphologies were isolated. Bacterial cellulolytic activity was tested biochemically. Marked gas production in the form of bubbles (0.1-4 cm) in Durham tubes was observed in H3, H7, H13, H15, H17, H21, and H22. Sugar degradation of all isolates was indicated by pink to maroon color development with the tetrazolium salt. Hallow zones (0.42-11 mm) by Congo red staining was exhibited by all strains except H2, H7, H8, and H19. Among the 26 bacterial isolates, 12 strains were identified as efficient cellulolytic bacteria. CMCase activity and ethanol titer of all isolates varied from 1.30 ± 0.03 (H13) to 1.83 ± 0.01 (H21) umol/mL/min and 2.36 ± 0.01 (H25) to 7.00 ± 0.01 (H21) g/L, respectively. Likewise, isolate H21 exhibited an ethanol yield of 0.40 ± 0.10 g/g with 78.38 ± 2.05% fermentation efficiency. Molecular characterization of four strains, Staphylococcus sp. H13, Acinetobacter baumanni H17, Acinetobacter sp. H21, and Acinetobacter nosocomialis H22, were based on the maximum cellulolytic index and the ethanol yield. H. indicola harbor promising and novel bacteria with a natural cellulolytic tendency for efficient bioconversion of LCB to value-added products. Hence, the selected cellulolytic bacteria can become an excellent addition for use in enzyme purification, composting, and production of biofuel at large.

Protective potential of fresh orange juice against zinc oxide nanoparticles-induced trans-placental and trans-generational toxicity in mice.

Due to the emerging applications of nanoparticles, human exposure to nanoparticles is unavoidable, particularly to zinc oxide nanoparticles (ZnO NPs), owing to their wide range of usage. The ongoing study aimed to evaluate trans-generational toxic potential of ZnO NPs through exposure to F0 mothers, in F1 pups and F1 mature offspring and the protective potential of fresh orange juice (OJ). Twenty-eight F0 mothers were randomly allocated into four groups (n = 7), control; untreated, dose group; exposed to ZnO NPs, dose+antidote group; coadministered ZnO NPs + OJ, antidote group; OJ, during the organogenetic period. Fifty percent of F0 mothers were subjected to cesarean sections on the 18th day of gestation and F1 pups were recovered, macro-photographed, and dissected for liver evisceration, while 50% of F0 mothers underwent standard delivery. After parturition, F1 offspring were examined, and the liver and blood samples were extracted. Observations showed that ZnO NPs exposure in F0 mothers in preparturition and postparturition resulted in decreased body weight, increased liver weight, and elevated levels of ALT and AST significantly p ≤ .05 as compared to the control and antidote groups. Histopathological analysis of maternal livers intoxicated with NPs showed the disruptive structure of central vein, hepatocytes, and Kupffer cells in F0 mothers, while F1 pups showed morphological deviations and distorted development of the liver tissue and congestion, in contrast to the control. F1 offspring of NPs exposed mothers, even at postnatal week 8 showed pyknotic nuclei and activated Kupffer cells in the liver sections against control. But in the case of the Dose+antidote group, alterations were less severe than in the dose group. It can be concluded that exposure to ZnO NPs instigates teratogenicity and hepatotoxicity in F1 pups, F0 mothers, and F1 offspring, respectively, while fresh orange juice acts as a remedial agent against the abovementioned toxicities.

Identification and in silico analysis of noval alteration Arg420Gly in KIT proto oncogene among acute myeloid leukemia patients.

A number of studies have reported frequent incidence of c-kit gene mutations in association with core binding factor acute myeloid leukemia (CBF-AML). These genetic changes have become important prognostic predictors in patients with abnormal karyotype. Aim of this study was the detection of nucleotide alterations in newly diagnosed acute myeloid leukemia patients for three exons of c-kit gene, including cytogenetically normal patients. Thirty-one de novo AML patients were screened for any possible variations in exon 8, 11 and 17 sequences of c-kit proto-oncogene leading to amino acid substitutions or frame shift. Sanger sequencing method was employed followed by sequence analysis. Mutation data was then correlated with clinical and hematological parameters of patients and prognostic significance of genetic changes was assessed as well. The computational tools were then used to further understand the extent of damage caused by these mutations to c-kit protein. Fifteen (48.4%) mutant patients were observed with single, double or multiple mutations in one, two or all three exons studied. The analysis revealed eight new alterations which were not reported previously. Significant variation among mutant and non-mutant group of patients was observed with respect to FAB subtypes (x2 = 12.524, p = 0.029), Spleen size (x2 = 4.288, p = 0.038) and Red blood cell count (x2 = 8.447, p = 0.007). The survival analysis indicates poor overall and event free survival outcomes in mutant individuals. Furthermore, the in silico analysis suggests that changes in nucleotide sequences can possibly damage the protein structure and effect it's function. This study emphasizes the need to consider screening of c-kit gene alterations not only in CBF-AML but in cytogenetically normal AML patients as well. In current investigation the effect of mutation Arg420Gly on structure and function of c-kit protein was investigated, as this was the most observed substitution in present cohort. Various bioinformatics tools and techniques were employed, which determined that Arg420Gly is possibly non-pathogenic mutation.

RUNX1 mutation and elevated FLT3 gene expression cooperates to induce inferior prognosis in cytogenetically normal acute myeloid leukemia patients.

BACKGROUND: Acute myeloid leukemia (AML) is a bone marrow malignancy having multiple molecular pathways driving its progress. In recent years, the main causes of AML considered all over the world are genetic variations in cancerous cells. The RUNX1 and FLT3 genes are necessary for the normal hematopoiesis and differentiation process of hematopoietic stem cells into mature blood cells, therefore they are the most common targets for point mutations resulting in AML. METHODS: We screened 32 CN-AML patients for FLT3-ITD (by Allele-specific PCR) and RUNX1 mutations (by Sanger sequencing). The FLT3 mRNA expression was assessed in all AML patients and its subgroups. RESULTS: Eight patients (25%) carried RUNX1 mutation (K83E) while three patients (9.37%) were found to have internal tandem duplications in FLT3 gene. The RUNX1 mutation data were correlated with clinical parameters and FLT3 gene expression profile. The RUNX1 mutations were observed to be significantly prevalent in older males. Moreover, RUNX1 and FLT3-mutated patients had lower complete remission rate, event-free survival rate, and lower overall survival rate than patients with wild-type RUNX1 and FLT3 gene. The RUNX1 and FLT3 mutant patients with up-regulated FLT3 gene expression showed even worse prognosis. Bradford Assay showed that protein concentration was down-regulated in RUNX1 and FLT3 mutants in comparison to RUNX1 and FLT3 wild-type groups. CONCLUSION: This study constitutes the first report from Pakistan reporting significant molecular mutation analysis of RUNX1 and FLT3 genes including FLT3 expression evaluation with follow-up. This provides an insight that aforementioned mutations are markers of poor prognosis but the study with a large AML cohort will be useful to further investigate their role in disease biology of AML.

Pomegranate peels waste hydrolyzate optimization by Response Surface Methodology for Bioethanol production.

Unwanted agricultural waste is largely comprised of lignocellulosic substrate which could be transformed into sugars. The production of bioethanol from garbage manifested an agreeable proposal towards waste management as well as energy causation. The goal of this work is to optimize parameters for generation of bioethanol through fermentation by different yeast strains while Saccharomyces cerevisiae used as standard strain. The low cost fermentable sugars from pomegranate peels waste (PPW) were obtained by hydrolysis with HNO3 (1 to 5%). The optimum levels of hydrolysis time and temperature were elucidated via RSM (CCD) ranging from 30 to 60 min and 50 to 100 °C respectively. The result shows that optimum values (g/L) for reducing sugars was 61.45 ± 0.01 while for total carbohydrates was 236 ± 0.01. These values were found when PPW was hydrolyzed with 3% HNO3, at 75 °C for one hour. The hydrolyzates obtained from the dilute HNO3 pretreated PPW yielded a maximum of 0.43 ± 0.04, 0.41 ± 0.03 g ethanol per g of reducing sugars by both Metchnikowia sp. Y31 and M. cibodasensis Y34 at day 7 of ethanologenic experiment. The current study exhibited that by fermentation of dilute HNO3 hydrolyzates of PPW could develop copious amount of ethanol by optimized conditions.

Punica granatum waste to ethanol valorisation employing optimized levels of saccharification and fermentation.

Pomegranate peels (PPW) as municipal waste is inexpensive biomass that could be a renewable source of sugars particularly rich in hemicellulosic contents. The subsequent conversion of available sugars in PPW can provide prospective strategy for cost-effective bioenergy production. In this study, an experimental setup based on CCD was implemented with the aim of bioconversion of biomass into bioethanol. The factors considered were Hydrochloric acid concentration (X1), the hydrolysis temperature (X2) and time (X3) for optimization with dilute Hydrochloric acid (HCl) saccharification. The present study investigates the optimised level of bioethanol synthesis from acid pre-treated PPW explained by RSM. Subsequently, three yeasts viz. Saccharomyces cerevisiae K7, Metschnikowia sp. Y31 and M. cibodasensis Y34 were utilized for fermentation of acid hydrolysed and detoxified feed stocks. Optimum values of reducing sugars 48.02 ± 0.02 (gL-1) and total carbohydrates 205.88 ± 0.13 (gL-1) were found when PPW was hydrolyzed with 1% HCl concentration at 100˚C of temperature for 30 min. Later on, fermentation of PPWH after detoxification with 2.5% activated charcoal. The significant ethanol (g ethanol/g of reducing sugars) yields after fermentation with Metschnikowia sp. Y31 and M. cibodasensis Y34 found to be 0.40 ± 0.03 on day 5 and 0.41 ± 0.02 on last day of experiment correspondingly. Saccharomyces cerevisiae K7 also produce maximum ethanol 0.40 ± 0.00 on last day of incubation utilizing the PPWH. The bioconversion of commonly available PPW into bioethanol as emphasize in this study could be a hopeful expectation and also cost-effective to meet today energy crisis.

Analysis of RAS gene mutations in cytogenetically normal de novo acute myeloid leukemia patients reveals some novel alterations.

Rat sarcoma gene (RAS) holds great importance in pathogenesis of acute myeloid leukemia (AML). The activated mutations in Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) and Kirsten rat sarcoma viral oncogene homolog (KRAS) confers proliferative and survival signals, deliberating numerous effects on overall survival and progression free survival in AML patients. In this study thirty one (31) blood samples of adult newly diagnosed AML patients were collected to identify possible incidence of mutations through amplification of KRAS (exon 1 and 2) and NRAS gene (exon 1 and 2) using polymerase chain reaction (PCR). Amplicons were then subjected to sequencing and were analyzed through Geneious Prime 2019. Five of thirty one (16.12%) patients had altered sites in either NRAS or KRAS. The NRAS mutations were observed in three AML patients (N = 3, 9.67%). A novel missense mutation NRAS-I36R (239 T > G) representing a substitution of single nucleotide basepair found in NRAS exon 1 while exon 2 was detected with heterozygous mutation NRAS-E63X (318G > T) and insertion (A), resulting in frameshift of the amino acid sequence and insertion of two nucleotide basepairs (TA) in two of the patients. KRAS mutations (N = 2, 6.45%) were found in exon 1 whereas no mutations in KRAS exon 2 were detected in our patient cohort. Mutation in KRAS Exon 1, KRAS-D30N (280G > A) was observed in two patients and one of them also had a novel heterozygous mutation KRAS-L16N (240G > C). In addition there was no statistically significant association of mutRAS gene of AML patients with several prognostic markers including age, gender, karyotyping, CD34 positivity, cytogenetic abnormalities, total leukocyte count, white blood cell count and French-American-British (FAB) classification. However, the presence of mutRAS gene were strongly associated (p = 0.001) with increased percentage of bone marrow blasts. The prevalence of mutations in correlation with clinical and hematological parameter is useful for risk stratification in AML patients.

Mycosynthesis of Size-Controlled Silver Nanoparticles through Optimization of Process Variables by Response Surface Methodology.

The present study was carried out to reduce the size of silver nanoparticles (AgNPs) by optimizing physico-chemical conditions of the Aspergillus fumigatus BTCB10 growth based on central composite design (CCD) through response surface methodology (RSM). Variables such as a concentration of silver nitrate (mM), NaCl (%) and the wet weight of biomass (g) were controlled to produce spherical, monodispersed particles of 33.23 nm size, observing 78.7% reduction in size as compared to the initially obtained size that was equal to 356 nm. The obtained AgNPs exhibited negative zeta potential of -9.91 mV with a peak at 420 nm in the UV-Vis range whereas Fourier Transform Infrared (FT-IR) analysis identified O-H, C = C, C ≡ C, C-Br and C-Cl groups attached as capping agents. After conducting RSM experiments, a high nitrate reductase activity value of 179.15 nmol/h/ml was obtained; thus indicating a likely correlation between enzyme production and AgNPs synthesis. The F-value (significant at 3.91), non-significant lack of fit and determination coefficient (R2 = 0.7786) is representative of the good relation between the predicted values of response. We conclude that CCD is an effective tool in obtaining significant results of high quality and efficiency.The present study was carried out to reduce the size of silver nanoparticles (AgNPs) by optimizing physico-chemical conditions of the Aspergillus fumigatus BTCB10 growth based on central composite design (CCD) through response surface methodology (RSM). Variables such as a concentration of silver nitrate (mM), NaCl (%) and the wet weight of biomass (g) were controlled to produce spherical, monodispersed particles of 33.23 nm size, observing 78.7% reduction in size as compared to the initially obtained size that was equal to 356 nm. The obtained AgNPs exhibited negative zeta potential of ­9.91 mV with a peak at 420 nm in the UV-Vis range whereas Fourier Transform Infrared (FT-IR) analysis identified O­H, C = C, C ≡ C, C­Br and C­Cl groups attached as capping agents. After conducting RSM experiments, a high nitrate reductase activity value of 179.15 nmol/h/ml was obtained; thus indicating a likely correlation between enzyme production and AgNPs synthesis. The F-value (significant at 3.91), non-significant lack of fit and determination coefficient (R2 = 0.7786) is representative of the good relation between the predicted values of response. We conclude that CCD is an effective tool in obtaining significant results of high quality and efficiency.

Detection of Exon 12 and 14 Mutations in Janus Kinase 2 Gene Including a Novel Mutant in V617F Negative Polycythemia Vera Patients from Pakistan.

The most frequently reported genetic aberration among polycythemia vera (PV) patients is a gain of function mutation V617F in exon 14 of Janus kinase 2 (JAK2) gene. However in many investigations, V617F negative PV patients have been reported to harbor mutations in JAK 2 exon 12. We investigated 24 patients with PV (diagnosed following 2016 WHO guidelines) to detect V617F mutation through allele specific PCR. The frequency of which was found to be 19/24 (79.2 %). Later on JAK2 exon 12 and 14 was amplified by conventional PCR in V617F negative patients and subjected to sequence analysis. A total of 03 mutated sites in exon 12 were detected in only two V617F-negative patients 2/5 (40%). All three substitutions were heterozygous i.e. F537F/I found in both patients and R528R/T, which is a novel mutation. In addition, one patient 1/5 (10%) manifested amino acid substitution V617A in JAK2 exon 14. Hematological parameters of individuals harboring mutations do not vary significantly than rest of the PV patients. Previous history and 2.3 years of follow-up studies reveal 15-year survival of V617F positive patients (n=19) to be 76%, while it is 94% for wild type V617 patients (n=05). Mean TLC of the patient cohort was 17.6± 9.1 x 109/L, mean platelet count was 552± 253 x 109/L, mean hemoglobin was 16.9± 3.2 g/dl, mean corpuscular volume (MCV) was 77.2± 13.0 fl and mean corpuscular hemoglobin (MCH) was 25.6± 3.9 pg. This is the very first attempt from Pakistan to screen JAK2-exon 12 mutations in PV patients. We further aim to investigate Jak2 exon 12 mutations in larger number of PV patients to assess their clinical relevance and role in disease onset, progression and transformation.

Screening of yeast isolates from flowers for effective ethanol production.

The use of alternative substrates to produce biofuel is a striking option nowadays. This study aimed to screen bioethanolproducing yeast strains. From different flowers, 65 yeasts were isolated. Twelve isolates assimilated glucose by liberation of CO 2 anaerobically. Out of these, only 5 yeast isolates fermented glucose in medium consisting of 0.8 g/L Mg2+ ions to produce 2.05 ± 0.03% ethanol. The selected five strains were identified as members of the genera Metschnikowia or Meyerozyma based on molecular characterization. Selected yeast strains were used for conversion of rice into bioethanol following dilute acid hydrolysis and fermentation. Consistent ethanol production was 1.80 ± 0.05% at days 2-4 by Metschnikowia cibodasensis Y34 and 2.20 ± 0.21% by Meyerozyma caribica Y42 at days 4-6 with a gradual decrease at the time of experiment termination (day 10). Metschnikowia cibodasensis Y34 and Meyerozyma caribica Y42 produced the highest ethanol at pH 3, i.e. 1.75 ± 0.14% at days 3 and 5 and 2.05 ± 0.14% at days 1 and 3, respectively, upon incubation with different pH levels and 1% NaCl. Growth and ethanol production at pH 4 and 5 was close to that at pH 3, with a slight increase in production by Metschnikowia cibodasensis Y34 at pH 4 up to day 3.