Sana Khurshid

Treatment of chronic wounds has been shifted to traditional approaches due to surge in antibiotic resistance. Wounds that fail to heal satisfactorily may result in the amputation of the organ. In this research work, cinnamon oil (CO) and aloe vera (AV) that have been traditionally used as antibacterial agents are combined in a unique gel (COVA) and its antibacterial activity has been evaluated through in vitro and in vivo studies. Antibacterial activity was measured through disk diffusion and agar dilution method against Pseudomonas aeruginosa and Staphylococcus aureus . To check antibacterial and wound healing activity, diabetic excision wound healing rat model was used. Wound closure, wound contraction, tissue hydroxyproline content, antioxidant capacity (TAC), and malondialdehyde (MDA) level were monitored. The minimum inhibitory concentrations of CO + AV for bacterium P. aeruginosa and S. aureus were 100 and 200 µg/ml, respectively. After 14 days, the wounds covered with COVA therapy reached to nearly full wound closure (79% wound contraction) compared to control. The collagen content and level of TAC increased significantly ( P < 0.05) in treated groups; therefore, 25% fast healing was observed in wounds treated with CO and AV gel combined. Reduced levels of tissue MDA were observed in all treated groups and specially wound covered with COVA (0.43 mM/mg in control vs 0.25 mM/mg in COVA). Histopathological examination also supported the outcomes. Significantly elevated increase in the level of hydroxyproline was found in rats of COVA treatment group (37.1 ± 0.44). Combination of CO and AV can be potentially used to prevent infection in wound; as these herbal agents not only inhibit the growth of pathogenic bacteria but also accelerate tissue repair.

Hepatocellular carcinoma (HCC) is a type of malignant tumor. miRNAs are noncoding RNAs and their differential expression patterns are observed in HCC-induced by alcoholism, HBV and HCV infections. By acting as a competing endogenous RNA (ceRNA), circRNA regulates the miRNA function, indirectly controlling the gene expression and leading to HCC progression. In the present study, data mining was performed to screen out all miRNAs and circRNA involved in alcohol, HBV or HCV-induced HCC with statistically significant (≤0.05%) expression levels reported in various studies. Further, the interaction of miRNAs and circRNA was also investigated to explore their role in HCC due to various causative agents. Together, these study data provide a deeper understanding of the circRNA–miRNA regulatory mechanisms in HCC. These screened circRNA, miRNA and their interactions can be used as prognostic biomarkers or therapeutic targets for the treatment of HCC.

Background: Gold nanoparticles have the potential to be used as a carrier in drug delivery system due to their small size, large surface area and short circulation time in blood. Objective: This study aims that doxorubicin conjugation with gold nanoparticles (AuNPs) may reduce its toxicity as well as improve therapeutic efficacy. Methods: Five groups of Albino rats were used; 1: healthy control, 2: Injured, 3: injured and treated with Dox, 4: Injured and treated with AuNPs, 5: Injured and treated with AuNPs: Dox. At the end of the experiment, blood and liver tissues were processed for biochemical and histopathological analysis. The expression of collagen, HO-1, IL-6 and TNF-α genes involved in liver fibrosis was observed through real-time PCR. Results: At the end of the experiment, it was observed that the body weights of DOX treated rats decreased by 0.72%, however, AuNPs and Au: DOX treated rats were 15.3% and 29.13% respectively. The percentage of liver protection determined through alanine aminotransferase and aspartate aminotransferase levels in DOX, AuNPs and AuNPs: DOX treated groups were 39.21%, 79.26%, 98.17% and 47.77%, 84.17%, 97.92% respectively. That represents better recovering liver in Dox-AuNPs treated rats compared to others. Histopathological and gene expression studies further support the findings. The mRNA expression levels of inflammatory and oxidative stress related genes HO-1, IL-6 and TNF-α were upregulated in the injured group but downregulated in the treated group. Conclusion: As depicted through biochemical, histopathological and gene expression studies, Au: DOX conjugate group seems to be protective against liver fibrosis.

Beta-Thalassemia (beta-thal), an autosomal recessive hemoglobinopathy, is one of the most common genetic disorders in Pakistan. Awareness of this disease, genetic counseling, extended family carrier screening and prenatal diagnosis (PND) are helpful in prevention and control. Currently, direct DNA sequencing and multiple amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) are the methods used to detect beta-thal mutations, the latter being the most widely used. This study aimed to evaluate PCR-high resolution melting (PCR-HRM) analysis for the detection of most common beta-thal mutations that are found in Pakistan. This study was designed to identify the beta-thal mutations using PCR-HRM analysis in a total of 90 samples [blood and chorionic villus sampling (CVS)]. These samples were first screened for routine mutations by MARMS-PCR and then evaluated by PCR-HRM analysis. The results of PCR-HRM analyses were further confirmed by direct DNA sequencing and all analyses interpreted the same results in all 90 samples. Eleven cases (36.6%) were detected to carry IVS-I-5 (G>C) (HBB: c0.92 + 5G>C), six cases (20.0%) with frameshift codons (FSC) 41/42 (-TTCT) (HBB: c.126_129delCTTT), five cases (16.0%) were diagnosed with codon 15 (G>A) (HBB: c.47G>A), three cases (10.0%) were found with codon 30 (G>C) (HBB: c.93G>C), one case was diagnosed with FSC 16 (-C) (HBB: c.51delC), one with IVS-I-1 (G>T) (HBB: c0.92 + 1G>T) and one with codon 5 (-CT) (HBB: c.17_18delCT). The PCR-HRM analysis represents a less tedious and more useful method for the detection of beta-globin gene mutations.

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum (C. thermocellum) by cloning in an Escherichia coli (E. coli) expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a K_m of 19.83 mM and a V_max of 0.12 μmol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C. thermocellum a potential candidate in industrial applications.

A simple extrusion method was used to entrap Synechocystis sp.P2A in alginate beads. The viability, growth response and Indoleacetic acid (IAA) production at different pH were studied in alginate immobilized Synechocystis sp.P2A. 2.6% sodium alginate (w/v) and pH-7 was found to be optimum for growth of Synechocystis sp. P2A as well as IAA production (79µg/ml). To prepare effective formulation for plant inoculation, alginate beads were further modified by coating with chitosan or chitosan-polyethylene glycol. Effect of all formulations containing Synechocystis sp. P2A in free and immobilized form on growth of Triticumaestivum was evaluated. Soil inoculation of entrapped Synechocystis in alginate beads coated with chitosan resulted in 20% increase in root length and 14% increase in dry weight as compared to non-inoculated seedlings. Free and immobilized cyanobacteria were allowed to grow in BG11 medium supplemented with 100μg/ml K2CrO4 and chromium reduction was measured at variable pH. At pH 7 immobilized showed 5% more reduction than free form. The current study showed that alginate immobilized Synechocystis sp. P2A can accomplish viable functions including plant growth promoting hormone production and chromium reduction and therefore propose an efficient and convenient method for storage and use of cyanobacteria.

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.