Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from Thermobifida fusca by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (Cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as Cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant Cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of Cel6A.CBC which revealed thermostability of Cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of Cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Assessment of polychlorinated biphenyls (PCBs) in the Himalayan Riverine Network of Azad Jammu and Kashmir.

The emission of polychlorinated biphenyls (PCBs) in South Asian countries is one of the great environmental concerns and has resulted in the contamination of surrounding high altitude regions such as Azad Jammu and Kashmir (AJK), Pakistan. This first investigation of Polychlorinated Biphenyl (PCBs) concentrations in the ambient air, water and surface soil was conducted along the extensive stream network in the AJK valley of the Himalayan Region. In 2014, surface soil samples were taken and passive air and water samplers were deployed along the four main rivers, namely Jhelum, Neelum, Poonch and Kunhar, and analysed for PCBs (33 congeners) using GC-MS/MS. The ∑33PCBs concentrations ranged from 31.17 to 175.2 (mean ±â€¯SD: 81 ±â€¯46.4 pg/L), ND to 1908 (1054 ±â€¯588.5 pg/g), and 29.8 to 94.4 (52.9 ±â€¯22.7 pg/m3) in surface water, soil and air matrices, respectively. The levels of dioxin-like PCBs (∑8DL-PCBs) contributed considerably towards the total PCBs concentrations: 60.63% (water), 43.87% (air) and 13.76% (soil). The log transformed air-water fugacity (log fa/fw) ratios ranged from -9.37 to 2.58; with 86.3% of the sampling sites showing net volatilization of selected PCB congeners. Similarly, the fugacity fractions for air-soil exchange exhibited narrow variation (0.8 to < 1) indicating net volatilization of PCBs. The ecological risk assessment showed low potential ecological risks (Eri  = 1.58-7.63) associated with PCB contamination. The present findings provide baseline data that suggest cold trapping of POPs in the remote mountainous areas of Pakistan and can support environmental management of POPs at the regional level. This pioneer investigation campaign to assess the PCBs concentrations in Himalayan Riverine Network of Azad Jammu and Kashmir, Pakistan helps to develop baseline data of PCBs from the strategically important riverine environment that would help in future regional as well as global ecological studies. However, the effects of temperature variations on the sampling rates of chemicals across a wide spectrum of volatility along the elevation gradient were not taken under consideration for PCBs atmospheric concentrations.

Silver nanoparticle impregnated chitosan-PEG hydrogel enhances wound healing in diabetes induced rabbits.

Non-healing wounds are among the serious complications of type-2-diabetes around the globe, associated with high incidence of bacterial infection, chronic nerve and blood vessel damage, and eventually repeated amputation of limbs and organs. Silver nanoparticles offer strong wound healing potential due to their well-known antibacterial activities. The present study reports the development of silver nanoparticle impregnated chitosan-poly ethylene glycol (PEG) hydrogel to accelerate wound healing in diabetic patients. The aim of the study was to formulate a sustained and slow release of silver nanoparticle using chitosan-PEG-Silver Nitrate based hydrogel for the treatment of chronic diabetic wounds. The silver nanoparticle containing chitosan-PEG pre-polymer solution was synthesized by reducing silver nitrate with PEG and chitosan solution, thereby, transforming the silver ions into silver nanoparticles. The resulted pre-polymer solution was then crosslinked using glutaraldehyde to form the desired hydrogel. The developed silver nanoparticle impregnated chitosan hydrogel was characterized using ultra-violet (UV) visible spectrophotometry, Fourier Transform-infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) followed by the determination of porosity, and swelling properties. The release of AgNPs from hydrogel was determined by UV-vis spectroscopy followed by antimicrobial and antioxidant assays. The wound healing efficacy of the synthesized hydrogel was evaluated in diabetic rabbits. The results demonstrated a higher porosity, higher degree of swelling and higher water vapor transition rate (WVTR) for silver nanoparticle impregnated hydrogel compared to bare chitosan-PEG hydrogel as well as improved antimicrobial and antioxidant properties in-vitro and enhanced wound healing capability in-vivo in diabetic rabbits. The hydrogel showed a slow and sustained release of AgNPs over a period of at least seven days manifesting the slow biodegradation of developed hydrogels. The improved antimicrobial, antioxidant and wound healing results indicate that the silver nanoparticle impregnated chitosan-PEG hydrogel can be a promising material for wound healing dressing for chronic diabetic wounds.

Assessment of organochlorine pesticides in the Himalayan riverine ecosystems from Pakistan using passive sampling techniques.

Organochlorine pesticides (OCPs) pose a considerable threat to human and environmental health. Despite most OCPs have been banned, they are still reported to be used in developing countries, including Pakistan. We aimed to identify the distribution, origin, mobility, and potential risks from OCPs in three major environmental compartments, i.e., air, water, and soil, across Azad Jammu and Kashmir valley, Pakistan. The sums of OCPs ranged between 66 and 530 pg/g in soil, 5 and 13 pg/L in surface water, and 14 and 191 pg/m3 in air, respectively. The highest sum of OCPs was observed in the downstream zone of a river that was predominantly influenced by peri-urban and urban areas. The OCP isomers ratios (α-HCH/γ-HCH and o,p'-DDT/p,p'-DDT) indicate use of lindane and technical DDTs mixture as a source of HCH and DDT in the riverine environment. Similarly, the ratios of DDE and DDD/the sum of DDTs, α-endosulfan/ß-endosulfan, and cis-chlordane/trans-chlordane indicate recent use of DDTs, endosulfan, and chlordane in the region. The air-water exchange fugacity ratios indicate net volatilization (fw/fa > 1) of α-endosulfan and trans-chlordane, and net deposition (fw/fa < 1) of ß-endosulfan, α-HCH, γ-HCH p,p'-DDD, p,p'-DDE, and p,p'-DDT. Based on the risk quotient (RQ) method, we consider the acute ecological risks for fish associated with the levels of OCPs as negligible. However, more studies are recommended to evaluate the chronic ecological risks to other riverine-associated aquatic and terrestrial species as well as human health risks to the POPs exposure through food chain transfer in forthcoming years.

Novel electrospun chitosan/polyvinyl alcohol/zinc oxide nanofibrous mats with antibacterial and antioxidant properties for diabetic wound healing.

Non-healing wound is a serious complication of diabetes, associated with extremely slow wound closure, and a high rate of infection, resulting in amputation or losses of limbs, high health care cost and poor quality of patient's life. In the present study, we hypothesized that nanofiber mats composed of a combination of chitosan, polyvinyl alcohol (PVA) and Zinc oxide (ZnO) could be an effective option for faster healing of diabetic wounds due to the wound healing activities of chitosan-PVA nanofibers and antibacterial properties of ZnO. Nanofiber mats of chitosan, PVA and ZnO were synthesized using electrospinning technique. The developed nanofibrous mats were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), antibacterial and antioxidant assays as well as in vivo wound healing experiments in rabbits. The results revealed that chitosan/PVA/ZnO nanofibrous membranes possessed higher antibacterial potential against E. coli, P. aeruginosa, B. subtilis and S. aureus compared to chitosan/PVA nanofibrous membranes. Moreover, chitosan/PVA/ZnO nanofibrous membranes exhibited higher antioxidant potential compared to chitosan/PVA nanofibrous mats. The in vivo wound healing studies showed that chitosan/PVA/ZnO nanofibrous membranes resulted in accelerated wound healing as compared to chitosan/PVA nanofibers. The current study, thus, reveals that chitosan/PVA/ZnO electrospun scaffolds could be effectively helpful in dressings for diabetic wounds.

mRNA secondary structure engineering of Thermobifida fusca endoglucanase (Cel6A) for enhanced expression in Escherichia coli.

The sequence and structure of mRNA plays an important role in solubility and expression of the translated protein. To divulge the role of mRNA secondary structure and its thermodynamics in the expression level of the recombinant endoglucanase in Escherichia coli, 5'-end of the mRNA was thermodynamically optimized. Molecular engineering was done by introducing two silent synonymous mutations at positions +5 (UCU with UCC) and +7 (UUC with UUU) of the 5'-end of mRNA to relieve hybridization with ribosomal binding site. Two variants of glycoside hydrolase family six endoglucanase, wild type (cel6A.wt) and mutant (cel6A.mut) from Thermobifida fusca were expressed and characterized in E. coli using T7 promoter-based expression vector; pET22b(+). Enhanced expression level of engineered construct (Cel6A.mut) with ∆G = -2.7 kcal mol(-1)was observed. It showed up to ~45 % higher expression as compared to the wild type construct (Cel6A.wt) having ∆G = -7.8 kcal mol(-1) and ~25 % expression to the total cell proteins. Heterologous protein was purified by heating the recombinant E. coli BL21 (DE3) CodonPlus at 60 °C. The optimum pH for enzyme activity was six and optimum temperature was 60 °C. Maximum activity was observed 4.5 Umg(-1) on CMC. Hydrolytic activity was also observed on insoluble substrates, i.e. RAC (2.8 Umg(-1)), alkali treated bagass (1.7 Umg(-1)), filter paper (1.2 Umg(-1)) and BMCC (0.3 Umg(-1)). Metal ions affect endoglucanase activity in different ways. Only Fe(2+) exhibited 20.8 % stimulatory effects on enzyme activity. Enzyme activity was profoundly inhibited by Hg2(+) (91.8 %).

Role of Ca2+ on growth of Brassica campestris L. and B. juncea (L.) Czern & Coss under Na+ stress.

Root and shoot growth of Brassica campestris L. and B. juncea increased significantly (P < 0.01) with enhanced Ca(2+) treatment along with 60 mM NaCl in the root medium. The maximum fresh mass of shoot and root in B. juncea was recorded at 10 mM Ca(2+) concentration. The relative growth rate of shoot of both species reached its maximum at 8 mM of Ca(2+) concentration. Average rate of Ca(2+) intake (I(Ca)) was higher in B. juncea than B. campestris. In B. juncea, the average transport of Ca(2+) to shoot increased by 19%, 38%, 119%, 125% and 169% compared with the control. Furthermore specific utilization rate of Ca(2+) was higher in B. juncea than B. campestris. In B. campestris it increased by 9%, 32%, 41% and 59% at 4, 6, 8, and 10 mM of calcium in comparison to 2 mM Ca(2+) treatment. At 4, 6, 8 and 10 mM of Ca(2+) application, the increase in the leaf area ratio was 10, 17, 23 and 30%, respectively. In the shoot and root portions of B. campestris and B. juncea, Ca(2+) had a linear relationship with potassium and sulfur, whereas it was in antagonism with sodium ion.

Detecting single-feature polymorphisms using oligonucleotide arrays and robustified projection pursuit.

MOTIVATION: Genomic DNA was hybridized to oligonucleotide microarrays to identify single-feature polymorphisms (SFP) for Arabidopsis, which has a genome size of approximately 130 Mb. However, that method does not work well for organisms such as barley, with a much larger 5200 Mb genome. In the present study, we demonstrate SFP detection using a small number of replicate datasets and complex RNA as a surrogate for barley DNA. To identify single probes defining SFPs in the data, we developed a method using robustified projection pursuit (RPP). This method first evaluates, for each probe set, the overall differentiation of signal intensities between two genotypes and then measures the contribution of the individual probes within the probe set to the overall differentiation. RESULTS: RNA from whole seedlings with and without dehydration stress provided 'present' calls for approximately 75% of probe sets. Using triplicated data, among the 5% of 'present' probe sets identified as most likely to contain at least one SFP probe, at least 80% are correctly predicted. This was determined by direct sequencing of PCR amplicons derived from barley genomic DNA. Using a 5 percentile cutoff, we defined 2007 SFP probes contained in 1684 probe sets by combining three parental genotype comparisons: Steptoe versus Morex, Morex versus Barke and Oregon Wolfe Barley Dominant versus Recessive. AVAILABILITY: The algorithm is available upon request from the corresponding author. CONTACT: xinping.cui@ucr.edu SUPPLEMENTARY INFORMATION: http://faculty.ucr.edu/~xpcui.

DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS.

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.