Characterization of a fungal α-galactosidase and its synergistic effect with ß-mannanase for hydrolysis of galactomannan.

An acid stable α-galactosidase was produced and purified from mannolytic fungal strain, Penicillium aculeatum APS1. Enzyme was produced using wheat bran and copra cake moistened with corn steep liquor by solid state fermentation. APS1αgal having molecular weight of 65.4 kDa was purified to electrophoretic homogeneity by three phase partitioning and gel permeation chromatography with high enzyme recovery. APS1αgal was found to be maximally active at 55 °C and pH 4.5, having high stability at acidic pH. Thermal stability and thermal inactivation kinetics of APS1αgal were also studied. APS1αgal was found to effectively hydrolyse oligosaccharides as well as polysaccharides having α-1,6 linked galactose. Abolishment of enzyme activity in N-brommosuccinimide revealed an important role of tryptophan residue in catalysis. APS1αgal had shown outstanding tolerance to NaCl and proteases. MALDI-TOF MS/MS analysis indicated that enzyme is probably a member of family GH27. Synergistic interaction between APS1αgal and ß-mannanase for hydrolysis of galactomannan was very clear and maximum 2.0° of synergy was found under simultaneous mode of action. This study reports a new source of α-galactosidase with biochemical properties suitable for applications in food and feed industries.

Bridge plating in the setting of radiocarpal instability: Does distal fixation to the second or third metacarpal matter? A cadaveric study.

BACKGROUND: Radiocarpal dislocations are rare but potentially devastating injuries. Poorer outcomes are associated with inadequate or lost reduction, such as ulnar translocation, but no consensus exists on the ideal fixation technique. Dorsal bridge plate fixation has been described for various settings in the treatment of complex distal radius fractures and can be fixed distally to the second or third metacarpal, but its application for radiocarpal dislocations has not been established. AIM: To determine whether distal fixation to the second or third metacarpal matters. METHODS: Using a cadaveric radiocarpal dislocation model, the effect of distal fixation was studied in two stages: (1) A pilot study that investigated the effect of distal fixation alone; and (2) a more refined study that investigated the effect of described techniques for distal and proximal fixation. Radiographs were measured in various parameters to determine the quality of the reduction achieved. RESULTS: The pilot study found that focusing on distal fixation alone without changing proximal fixation results in ulnar translocation and volar subluxation when fixing distally to the second metacarpal compared with the third. The second iteration demonstrated that anatomic alignment in coronal and sagittal planes could be achieved with each technique. CONCLUSION: In a cadaveric radiocarpal dislocation model, anatomic alignment can be maintained with bridge plate fixation to the second metacarpal or the third metacarpal if the described technique is followed. When considering dorsal bridge plate fixation for radiocarpal dislocations, the surgeon is encouraged to understand the nuances of different fixation techniques and how implant design features may influence proximal placement.

Thermotolerant and protease-resistant GH5 family ß-mannanase with CBM1 from Penicillium aculeatum APS1: purification and characterization.

In past several years, mannanases has attracted many researchers owing to its extensive industrial applications. The search for novel mannanases with high stability still continues. Present investigation was focused on purification of extracellular ß-mannanase from Penicillium aculeatum APS1 and its characterization. APS1 mannanase was purified to homogeneity by chromatography techniques. Protein identification by MALDI-TOF MS/MS revealed that the enzyme belongs to GH family 5 and subfamily 7, and possesses CBM1. The molecular weight was found to be 40.6 kDa. The optimum temperature and pH of APS1 mannanase were 70 °C and 5.5, respectively. APS1 mannanase was found to be highly stable at 50 °C and tolerant at 55-60 °C. The enzyme was very sensitive to Mn+2, Hg+2 and Co+2 metal ions and stimulated by Zn+2. Inhibition of activity by N-bromosuccinimide suggested key role of tryptophan residues for catalytic activity. The purified enzyme was efficient in hydrolysis of locust bean gum, guar gum and konjac gum and kinetic studies revealed highest affinity towards locust bean gum (LBG). APS1 mannanase was found to be protease resistant. Looking at the properties, APS1 mannanase can be a valuable candidate for applications in bioconversion of mannan-rich substrates into value-added products and also in food and feed processing.

Impact of Blood Transfusion in Free Flap Breast Reconstruction Using Propensity Score Matching.

BACKGROUND: Microvascular thrombosis has been associated with cytokine release and inflammatory syndromes which can occur as a result of blood transfusions. This phenomenon could potentially lead to complications in breast free flap reconstruction. The aim of this study was to evaluate the impact of perioperative blood transfusion in free flap breast reconstruction using large population analysis. METHODS: The American College of Surgeons National Quality Improvement Program database was queried for delayed free flap breast reconstructions performed in 2016. The study population was divided based on perioperative blood transfusion within 24 hours of the start of the operation. Propensity score matching analysis was used to ensure homogeneity between the two study groups. Primary outcome was unplanned return to the operating room (OR) within 30 days. Secondary outcomes were readmission and complications. RESULTS: A total of 1,256 patients were identified. Out of those, 91 patients received a perioperative blood transfusion. All the patients received only one unit of PRBC within the first 24 hours. Those patients were matched with similar patients who did not receive a transfusion on a ratio of 1:3 (273 patients). Patients who received a transfusion had a significantly higher incidence of reoperation (42 vs. 10%, p < 0.001). Patients who received a transfusion were more likely to return to the OR after 48 hours from the initial operation (13 vs. 5%, p = 0.001). All returns to the OR were due to flap-related complications. Perioperative blood transfusion increased the incidence of wound dehiscence (9 vs. 2%, p = 0.041) but had no protective effect on the development of other postoperative complications. CONCLUSION: Perioperative blood transfusion in free flap breast reconstruction is associated with an increased probability of flap-related complications and subsequent return to the OR without decreasing the probability of developing other systemic postoperative complications.

Perioperative Fluid Resuscitation in Free Flap Breast Reconstruction: When Is Enough Enough?

Perioperative liberal fluid resuscitation (LFR) can result in interstitial edema and venous congestion and may be associated with compromised perfusion of free flaps and higher incidence of wound complications. We hypothesized that restrictive intraoperative fluid resuscitation improves flap perfusion and lowers the wound complication rate in free flap breast reconstruction. METHODS: Patients undergoing free flap reconstruction of the breast from 2010 to 2018 were retrospectively reviewed. The study population was divided into 2 groups, LFR (≥7 ml/kg/h) and restrictive fluid resuscitation (RFR) (<7 ml/kg/h). Mean percutaneous oximetry readings of the flap over the first 24 hours were recorded. Primary outcome was development of any wound complication. Secondary outcomes were mean percutaneous oximetry readings within the first 24 hours, length of stay, and development of acute kidney injury. RESULTS: One hundred twenty-six patients were identified, with 41 patients undergoing LFR. The mean fluid received for the RFR group was 5.5 versus 10.2 ml/kg/h for the LFR group (P < 0.001). LFR resulted in a significantly higher incidence of wound complications (76% versus 15%, P < 0.001). The mean oximetry readings within 24 hours were significantly lower for the LFR group (41% versus 56%, P < 0.001). Urine output intraoperatively and within the first 24 hours was similar between the 2 groups. No patients developed acute kidney injury. CONCLUSION: RFR in free flap breast reconstruction is associated with increased flap perfusion and lower incidence of wound-related complications and should be considered whenever possible.

Clostridium difficile Infection in the Plastic Surgery Population: Lessons from the ACS NSQIP Database.

Clostridium difficile-associated infections (CDI) have a significant impact on morbidity and mortality of hospitalized medical and surgical patients. There is a paucity of data regarding the incidence, impact, and modifiable risk factors in the plastic surgery population. METHODS: The ACS NSQIP database was retrospectively queried for all cases performed by plastic surgeons during 2016. All plastic surgery cases, combined cases, demographics, and baseline clinical characteristics were extracted from the database. The study population was divided into 2 groups based on the development of CDI. Independent variables for development of CDI were identified. RESULTS: During the study period, a total of 29,256 patients underwent a procedure by plastic surgery, with the most commonly performed procedures involving the breast (58%) and trunk (14%). Only 44 patients developed post-operative CDI (0.1%). Factors independently associated with development of CDI were wound classification at the end of the surgery, COPD, procedures involving the trunk, and surgery for reconstruction of pressure ulcers. Outpatient surgery was associated with decreased odds of developing CDI [AOR (95% CI):0.2 (0.1, 0.4), adj P < 0.001]. Staying overnight did not increase the odds of developing CDI; however, staying for >1 day in the hospital was associated with an increased risk of CDI development [AOR (95% CI): 1.03 (1.01, 1.13), adj P = 0.001]. Combined cases, ASA, body mass index, diabetes, and active smoking were not associated with CDI. CONCLUSIONS: CDI are rare in the plastic surgery population and are most associated with trunk/decubitus ulcer reconstructions, inpatient hospital stay, and contaminated wounds. The patients that usually fit in these categories have acutely or chronically infected wounds, which are often treated with systemic antibiotics. For patient with decubitus ulcers and other trunk reconstruction, the guidelines for pre and post-operative systemic antibiotic usage is not well defined. For patients who have had trunk reconstruction, development of evidence-based antibiotic stewardship guidelines may help these patients by limiting antibiotic usage and thereby reducing the incidence of CDI.

Effect of internal mammary vessels radiation dose on outcomes of free flap breast reconstruction.

To assess the impact of internal mammary (IM) vessels radiation dose on autologous free-flap based breast reconstruction outcomes. We retrospectively evaluated the medical records of breast cancer patients who underwent mastectomy and free-flap breast reconstruction after postoperative radiation therapy (RT) to the breast/chest wall with (n = 9) or without (n = 11) electively including the IM lymph nodes. Twenty patients were included. Median age at diagnosis was 50 years (range, 33-63). The median time interval between the start of RT and reconstructive surgery was 16 months (range, 6-45). The maximal IM vessels dose was not associated with the risk of all complications (P = 0.44) or fat necrosis (P = 0.31). The mean IM vessels dose was not significant for the risk of all complications (P = 0.13) but was significant for fat necrosis (P = 0.04). A high mean IM vessels dose was related to the occurrence of fat necrosis.

Factors Affecting Women's Success in Academic and Private Practice Plastic Surgery.

Because plastic surgery trainees generally spend the majority of their training in academic centers and may have minimal exposure to other practice models, it can be difficult to decide which is the best route to achieve satisfaction and success. Surgeons need to be aware of benefits and challenges associated with different practice models and with workplace factors in general that contribute to happiness at work. This article reviews common practice models and provide specific and practical advice for the female surgeon.

Pediatric Burn Care: Unique Considerations in Management.

Severe pediatric burns require a multidisciplinary team approach at a specialized pediatric burn center. Special attention must be paid to estimations of total body surface area, fluid resuscitation and metabolic demands, and adequate analgesia and sedation. Long-term effects involve scar management and psychosocial support to the child and their family. Compassionate comprehensive burn care is accomplished by a multidisciplinary team offering healing in the acute setting and preparing the child and family for long-term treatment and care.

Manipulation of Human Primary Endothelial Cell and Osteoblast Coculture Ratios to Augment Vasculogenesis and Mineralization.

Tissue-engineering scaffolds are often seeded with a single type of cell, but there has been more focus on cocultures to improve angiogenesis and bone formation for craniofacial applications. Investigation of bone-derived osteoblasts (OBs) is important because of the use of bone grafts and migration of OBs from native bone into constructs in vivo and therefore, their contribution to bone formation in vivo. The limitation of primary OBs has been their inability to mineralize without osteogenic factors in vitro. Through coculture of OBs and endothelial cells (ECs) and manipulation of the coculture ratio, mineralization can be achieved without osteogenic media or additional growth factors, thus enhancing their utility for tissue-engineering applications. An optimal ratio of EC/OB for vasculogenesis and mineralization has not been determined for human primary cells. Human umbilical vein ECs were cultured with normal human primary OBs in different EC/OB ratios, namely, 10:1, 5:1, 1:1, 1:5, and 1:10 with EC and OB monocultures as controls. The number of vasculogenic networks in a collagen matrix was highest in ratios of 5:1 and 1:1. ECs lined up and formed capillary-like networks by day 10, which was not seen in the other groups. On polystyrene, cells were cocultured with ECs and OBs in direct contact (direct coculture) or separated by a transwell membrane (indirect coculture). At day 21, Alizarin Red staining showed mineralization on the 1:5 and 1:10 direct coculture ratios, with 1:5 having more mineralization nodules present than 1:10. No mineralization was seen in other direct coculture ratios or in any of the indirect coculture ratios. Alkaline phosphatase secretion was highest in the 1:5 direct coculture group. Vascular endothelial growth factor secretion from OBs was present in the 1:5 and 1:10 direct coculture ratios at all time points and inhibited after day 1 in other coculture groups. To improve vasculogenesis, cocultures of primary human ECs and OBs in ratios of 5:1 should be used, but to improve bone formation, the 1:5 direct coculture ratio results in most mineralization.

An efficient and cost-effective method for DNA extraction from athalassohaline soil using a newly formulated cell extraction buffer.

The present study describes the rapid and efficient indirect lysis method for environmental DNA extraction from athalassohaline soil by newly formulated cell extraction buffer. The available methods are mostly based on direct lysis which leads to DNA shearing and co-extraction of extra cellular DNA that influences the community and functional analysis. Moreover, during extraction of DNA by direct lysis from athalassohaline soil, it was observed that, upon addition of poly ethylene glycol (PEG), isopropanol or absolute ethanol for precipitation of DNA, salt precipitates out and affecting DNA yield significantly. Therefore, indirect lysis method was optimized for extraction of environmental DNA from such soil containing high salts and low microbial biomass (CFU 4.3 × 104 per gram soil) using newly formulated cell extraction buffer in combination with low and high speed centrifugation. The cell extraction buffer composition and its concentration were optimized and PEG 8000 (1 %; w/v) and 1 M NaCl gave maximum cell mass for DNA extraction. The cell extraction efficiency was assessed with acridine orange staining of soil samples before and after cell extraction. The efficiency, reproducibility and purity of extracted DNA by newly developed procedure were compared with previously recognized methods and kits having different protocols including indirect lysis. The extracted environmental DNA showed better yield (5.6 ± 0.7 µg g-1) along with high purity ratios. The purity of DNA was validated by assessing its usability in various molecular techniques like restriction enzyme digestion, amplification of 16S rRNA gene using PCR and UV-Visible spectroscopy analysis.

Differentiated adipose-derived stem cell cocultures for bone regeneration in polymer scaffolds in vivo.

Critical-sized bone defects can lead to significant morbidity, and interventions are limited by the availability and donor-site morbidity of bone grafts. Polymer scaffolds seeded with cells have been explored to replace bone grafts. Adipose-derived stem cells have shown great promise for vascularization and osteogenesis of these constructs, and cocultures of differentiated stem cells are being explored to augment vessel and bone formation. Adipose-derived stem cells were differentiated into endothelial cells and osteoblasts, and in vitro studies showed increased proliferation of cocultured cells compared with undifferentiated adipose-derived stem cells and monocultures of endothelial cells and osteoblasts. The cells were seeded into polylactic acid gas-plasma-treated scaffolds as cocultures and monocultures and then implanted into critical-sized rat calvarial defects. The cocultures were in a 1:1 osteoblast to endothelial cell ratio. The increase in proliferation seen by the cocultured cells in vitro did not translate to increased vascularization and osteogenesis in vivo. In vivo, there were trends of increased vascularization in the endothelial cell group and increased osteogenesis in the osteoblast and endothelial monoculture groups, but no increase was seen in the coculture group compared with the undifferentiated adipose-derived stem cells. Endothelial cells enhance vascularization and osteoblast and endothelial cell monocultures enhance bone formation in the polymer scaffold. Predifferentiation of adipose-derived stem cells is promising for improving vascularization and osteogenesis in polymer scaffolds but requires future evaluation of coculture ratios to fully characterize this response.

Enhanced conjugation of Candida rugosa lipase onto multiwalled carbon nanotubes using reverse micelles as attachment medium and application in nonaqueous biocatalysis.

Three liquid phases (viz. aqueous, nonaqueous, and reverse micelles) were scrutinized as medium for attachment of the enzyme Candida rugosa lipase (CRL) onto multiwalled carbon nanotubes (CNTs). The nanotubes were functionalized to attain carboxyl and amino groups on their surfaces before enzyme conjugation. Transmission electron microscopy and Fourier transformation infrared spectroscopic studies were used for characterization of the nanotubes during the course of functionalization. High enzyme loadings associated with the functionalized CNTs were observed when reverse micelles were used as the attachment medium. In addition, high activity in terms of ester synthesis in organic solvents was also observed while using those preparations. The nanobioconjugates prepared using reverse micelles were found to be highly sturdy and exhibited appreciable operational stability of around 95 ± 3% at 20th cycle (in case of carboxylated nanotubes) and 90 ± 5% at 10th cycle (in case of aminated nanotubes) for esterification. This shows the potential application of reverse micelles as the attachment medium for surface active enzymes such as CRL onto CNTs.

Performance evaluation of anaerobic hybrid reactors with different packing media for treating wastewater of mild alkali treated rice straw in ethanol fermentation process.

Four anaerobic hybrid reactors with different packing media viz. gravel (R1), pumice stone (R2), polypropylene saddles (R3) and ceramic saddles (R4) were operated in semi-continuous mode. Biomethanation potential of the wastewater generated during alkali-treatment of rice straw in ethanol production process was investigated at ambient conditions. The reactors were operated with varying organic loading rates (0.861-4.313 g COD l(-1) d(-1)) and hydraulic retention time (3-15 days). Higher COD removal efficiency (69.2%) and methane yield (0.153 l CH4 g(-1) CODadded) were achieved in reactor R2 at 15 days HRT. Modified Stover-Kincannon model was applied to estimate the bio-kinetic coefficients and fitness of the model was checked by the regression coefficient for all the reactors. The model showed an excellent correlation between the experimental and predicted values. The present study demonstrated the treatment of wastewater from alkali treated rice straw for production of biogas.

Robust nanobioconjugates of Candida antarctica lipase B--multiwalled carbon nanotubes: characterization and application for multiple usages in non-aqueous biocatalysis.

Present work elucidates two approaches for covalent attachment of the enzyme Candida antarctica lipase B (CALB) on multiwalled carbon nanotubes (MWCNTs). One method of enzyme immobilization involved carbodiimide chemistry while in the other approach, the cross linker (3-Aminopropyl) triethoxysilane (APTES) followed by succinic acid anhydride (SAA) were employed prior to carbodiimide activation. Modified MWCNTs were characterized by transmission electron microscopy (TEM), Fourier transformation infrared spectroscopic (FTIR), Raman spectroscopy and thermal gravitometric analysis (TGA). The lipase-MWCNTs conjugates were applied for synthesis of the flavor ester 'pentyl valerate' in cyclohexane and effects of solvent, temperature and agitation on ester synthesis were studied. Upon subject to reusability studies for 50 cycles, the bionanoconjugates were found to be highly sturdy and exhibited ≈ 79% activity (immobilization using carbodiimide) whereas the nanoconjugate prepared using APTES and SAA retained only up to ≈ 30% activity.

Production of cellulases by solid state fermentation with Aspergillus terreus and enzymatic hydrolysis of mild alkali-treated rice straw.

Rice straw was used as substrate for cellulase production by solid state fermentation with Aspergillus terreus. Substrate concentration, moisture ratio, inoculum size and initial pH were optimized using response surface methodology involving Box-Behnken design. The predicted filter paper activity under optimized parameters was 9.73 U/g and the validated filter paper activity was 10.96 U/g. Hydrolysis of the biomass pretreated with 0.125% to 1% NaOH for 24h at room temperature was performed using crude cellulase preparation. Treatment with 0.5% NaOH at room temperature for 24h was the most efficient treatment method for saccharification. Under the optimized conditions, rice straw yielded 676 mg reducing sugars per gram of substrate at a cellulase loading of 9 FPU g(-1) substrate. The present study establishes the possibility of using mild alkali pretreated rice straw for the production of fermentable sugars with 74.19% efficiency which can be further utilized for the production of ethanol.

Effect of endothelial differentiated adipose-derived stem cells on vascularity and osteogenesis in poly(D,L-lactide) scaffolds in vivo.

Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro-computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm(3), respectively, were statistically greater than that for ASC-Endo, 0.28 mm(3) (P < 0.05). There was no statistical difference between the PLA control (0.5 mm(3)) and ASC-Endo (0.28 mm(3)) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.

Migration of co-cultured endothelial cells and osteoblasts in composite hydroxyapatite/polylactic acid scaffolds.

Regeneration of bone in large segmental bone defects requires regeneration of both cortical bone and trabecular bone. A scaffold design consisting of a hydroxyapatite (HA) ring surrounding a polylactic acid (PLA) core simulates the structure of bone and provides an environment for indirect and direct co-culture conditions. In this experiment, human umbilical vein endothelial cells (EC) and normal human primary osteoblasts (OB) were co-cultured to evaluate cell migration and interactions within this biphasic composite scaffold. Both cell types were able to migrate between the different material phases of the scaffold. It was also observed that OB migration increased when they were co-cultured with ECs, whereas EC migration decreased in co-culture. The results show that co-culture of ECs and OBs in this composite biphasic scaffold allows for migration of cells throughout the scaffold and that pre-seeding a scaffold with ECs can increase OB infiltration into desired areas of the scaffold.

Improvement of the quality of whole wheat bread by supplementation of xylanase from Aspergillus foetidus.

The possible use of xylanase from Aspergillus foetidus MTCC 4898 as a bread improver was tested in whole wheat bread. The partially purified xylanase was used as an additive at 12 U/g during mixing of wheat flour. The effects of xylanase addition on the fermentation stage and the final bread quality were analyzed. Remarkable decrease (11%) in water absorption and increase in dough rising (28.5%) were noticed. Final moisture content of the bread was higher (40.5%) than control (32.3%). Improvements in volume (53%) and specific volume (56%) were also significant. Sensory evaluation indicated better flavour, taste, softness and overall acceptability. Texture profile analysis confirmed the rheological changes. Firmness was decreased by more than four folds. Improvements in cohesiveness and decline in springiness and gumminess were observed.