Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate-binding module.

The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate-binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N-terminus, native form (CelO-BC, 62 kDa); catalytic domain only (CelO-C, 42 kDa); CBM at the C-terminus (CelO-CB, 54 kDa) and CBM attached at both termini (CelO-BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO-C, CelO-CB, CelO-BC, and CelO-BCB were 35%, 35%, 30%, and 20%, respectively. The enzyme activities of CelO-C, CelO-CB, CelO-BC, and CelO-BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per µmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO-C, CelO-CB, CelO-BC, and CelO-BCB showed KM values of 32, 33, 45, and 43 mg⋅mL-1 and Vmax values of 3571, 3846, 3571, and 4545 U⋅min-1 , respectively. CBM3b at the N-terminus of GH5 linked through a P/T-rich linker was found to enhance the catalytic activity and thermostability of the enzyme.

Psychological Stress and Satisfaction with Life among Physical Therapy Students of Rawalpindi / Islamabad, Pakistan.

OBJECTIVE: To determine the occurrence of perceived stress and satisfaction with life as well as the association between the two among under graduate physical therapy students. METHODS: The cross-sectional study was conducted from January to July 2015 at Riphah International University, Isra University and Foundation University, Islamabad, Pakistan, and comprised Doctor of Physical Therapy students aged 17-23 years. Perceived Stress Scale and Satisfaction with Life Scale were used as tools to measure the outcomes. Data was analysed using S P SS 2 0.. RESULTS: Of the 306 students approached, 279(91%) responded. There were 39(14%) males and 240(86%) females with an overall mean age of 19.18±0.92 years. Mean stress and satisfaction with life scores were 21.35±5.76 and 24.49±5.77. Significant negative co-relation between the two scores was observed (p=0.001). There was no significant association of age with satisfaction with life (p=0.591) and perceived stress (p=0.283). A significant gender-based difference was observed in terms of perceived stress (p=0.037) and satisfaction with life ( (p=0.008), with females scoring higher. CONCLUSIONS: Male students were more satisfied with life compared to female students, and satisfaction with life decreased with an increase in stress.

Ultrasonographic evaluation of the ankle after unilateral traumatic lower limb amputations.

BACKGROUND: Unilateral lower limb amputation can alter the tissue morphology leading to increase future risk of degenerative secondary disorders. METHODS: Thirty-four consecutive male patients with unilateral lower limb amputations of different levels and 34 well matched male controls were included. To explore whether the ankles of traumatic lower limb amputees were undergoing morphological changes, three different tissue types i.e. talar cartilage, plantar fascia and Achilles tendon thicknesses in the intact limb of the lower limb amputee and healthy controls were measured by using ultrasound. RESULTS: Plantar fascia was found to be thicker (p=0.013) and talar cartilage was thinner (p<0.001) on the intact sides of the patients than those of the controls. Achilles tendon thickness was found to be similar. In patients group, plantar fascia thickness was positively correlated with age (r=0.601, p<0.001), BMI (r=0.454, p=0.007) and durations of amputation (r=0.443, p=0.009) and prosthetic use (r=0.429, p=0.011). Achilles tendon thickness was positively correlated with durations of amputation (r=0.338, p=0.05) and prosthetic use (r=0.468, p=0.005). In controls group, talar cartilage thickness was negatively correlated with age (r=-0.640, p<0.001) and BMI (r=-0.401, p=0.019). CONCLUSIONS: The talar cartilage seemed to be thinner and the plantar fascia to be thicker on the intact sides of the unilateral limb amputees.

Autologous mesenchymal stromal cell transplantation for spinal cord injury: A Phase I pilot study.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) transplantation has immerged as promising therapeutic approach to treat spinal cord injury (SCI). In this pilot study, we investigated the safety of intrathecal injection of autologous bone marrow-derived MSCs in nine patients with SCI. METHODS: Patients with complete SCI at the thoracic level were divided into two groups: chronic (>6 months, group 1) and sub-acute SCI (<6 months, group 2), according to time elapsed since injury. MSCs were isolated by density gradient separation of autologous bone marrow harvested from the iliac crest. Cells were cultured in a Good Manufacturing Practice-compliant facility to produce clinical scale dose. After quality control testing, MSCs were injected back to patients by intrathecal injection. Safety was defined as absence of adverse event and side effects after 1 month after receiving the injection. RESULTS: Six patients had chronic SCI with a median duration of 33 months since date of injury (range: 10-55 months), and three patients were in sub-acute phase of disease. Each patient received two or three injections with a median of 1.2 × 10(6) MSCs/kg body weight. No treatment-related adverse event was observed during median follow-up of 720 days (range: 630-826 days) in group 1 and 366 days (range: 269-367 days) in group 2, respectively. DISCUSSION: This pilot study demonstrated that autologous MSCs can be safely administered through intrathecal injection in spinal cord injury patients. Further investigation through randomized, placebo-controlled trials is needed.

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 %).

A non-ionic surfactant reduces the induction time and enhances expression levels of bubaline somatotropin in Pichia pastoris.

This study describes a simple approach for enhanced secretory expression of bubaline somatotropin (BbST) in the methylotropic yeast Pichia pastoris. A Mut(s) Pichia transformant carrying multi-copy, non-codon optimized BbST cDNA sequence, expressed and secreted the recombinant protein into the culture medium to a level of 25 % of the total proteins in the culture supernatant, after 120 h of induction. Inclusion of polysorbate-80 in the inducing medium resulted in a significant improvement in the BbST expression (up to 45 % of the total culture supernatant proteins) with concomitant reduction in the induction time to 48 h. The amount of BbST obtained was 148 mg/L, which was around fivefold higher than that obtained without the surfactant. BbST was purified to near homogeneity by FPLC on Q-sepharose FF anion-exchange column. Protein authenticity was judged by SDS-PAGE and western blot analyses. A bioassay based on proliferation of Nb2 rat lymphoma cell lines confirmed that the purified, recombinant BbST is biologically active. Use of polysorbate-80 in combination with methanol, during the induction phase, is likely to have general applicability in lowering the induction time and enhancing the secretory expression of other commercially important proteins in Mut(s) strains of P. pastoris.

Truncation of PstS1 antigen of Mycobacterium tuberculosis improves diagnostic efficiency.

PstS1, also named 38-kDa antigen, is one of the earliest known immune-dominant antigens of Mycobacterium tuberculosis and it has been commonly used in serodiagnostic tests. We constructed a truncated version, tnPstS1, by removing 96 and 14 amino acid residues from the N- and C-terminals, respectively of the native PstS1. The native and the truncated 29.5 kDa proteins were expressed in insoluble forms in Escherichia coli to levels of 15% and 25% of the total cell proteins, respectively. Both the variant molecules reacted equally well with the antisera raised in rabbit against the native protein. PstS1 and tnPstS1 were evaluated through ELISA against plasma samples from 160 culture positive tuberculosis patients and 40 healthy controls. With tnPstS1 43% of the patient samples were detected positive for the antibody as compared to only 36% in the case of the native PstS1. Data for the secondary structures of the native and the truncated variants as obtained by circular dichroism agreed with the known 3-D structure of the native protein and the predicted structure of the truncated version, respectively. The results show that the truncated tnPstS1 is more efficient as compared to the native PstS1 for use as a serodiagnostic agent.

Production enhancement and refolding of caprine growth hormone expressed in Escherichia coli.

This study describes comparison between IPTG and lactose induction on expression of caprine growth hormone (cGH), enhancing cell densities of Escherichia coli cultures and refolding the recombinant cGH, produced as inclusion bodies, to biologically active state. 2-3 times higher cell densities were obtained in shake flask cultures when induction was done with lactose showing almost same level of expression as in case of IPTG induction. With lactose induction highest cell densities were achieved in TB (OD(600) 16.3) and M9NG (OD(600) 16.1) media, producing 885 and 892 mg cGH per liter of the culture, respectively. Lactose induction done at mid-exponential stage resulted in a higher cell density and thus higher product yield. cGH over-expressed as inclusion bodies was solubilized in 50 mM Tris-Cl buffer (pH 12.5) containing 2 M urea, followed by dilution and lowering the pH in a step-wise manner to obtain the final solution in 50mM Tris-Cl (pH 9.5). The cGH was purified by Q-Sepharose chromatography followed by gel filtration with a recovery yield of 39% on the basis of total cell proteins. The product thus obtained showed a single band by SDS-PAGE analysis. MALDI-TOF analysis showed a single peak with a mass of 21,851 dalton, which is very close to its calculated molecular weight. A bioassay based on proliferation of Nb2 rat lymphoma cells showed that the purified cGH was biologically active.

Pressure ulcers in spinal cord injury: an unusual site and etiology.

Case series of pressure ulcers in patients with spinal cord injury after inappropriate and prolonged application of graduated compression stockings are presented. Three adult patients with spinal cord injury were fitted with graduated compression stockings in the acute phase to prevent thromboembolism. Acute hospital staff did not monitor the skin for potential pressure ulcer because of the graduated compression stockings. Grade II pressure ulcers developed on the toes in all three cases. All pressure ulcers were managed conservatively and healed without complication. The manufacturers of graduated compression stockings did not include information about the need to monitor patients with sensory impairment for pressure ulcers. It is suggested that the manufacturers of graduated compression stockings include information about the need to monitor patients with sensory impairment for skin breakdown. When graduated compression stockings are applied to patients with sensory impairment, including spinal cord injury, staff needs to be vigilant for pressure ulcers occurring at the extremes of the graduated compression stockings, in addition to the more common areas where pressure ulcers occur.