Renal transplant recipients undergoing endovascular abdominal aortic aneurysm repair have increased risk of perioperative acute kidney injury but no difference in late mortality.

OBJECTIVE: Renal transplant is associated with substantial survival advantage in patients with end-stage renal disease. However, little is known about the outcomes of renal transplant recipients (RTRs) after endovascular abdominal aortic aneurysm repair (EVAR). This study aimed to study the effect of renal transplant on perioperative outcomes and long-term survival after elective infrarenal EVAR. METHODS: The Vascular Quality Initiative database was queried for all patients undergoing elective EVAR from 2003 to 2021. Functioning RTRs were compared with non-renal transplant recipients without a diagnosis of end-stage renal disease (non-RTRs). The outcomes included 30-day mortality, acute kidney injury (AKI), new renal failure requiring renal replacement therapy (RRT), endoleak, aortic-related reintervention, major adverse cardiac events, and 5-year survival. A logistic regression analysis was used to assess the association between RTRs and perioperative outcomes. RESULTS: Of 60,522 patients undergoing elective EVAR, 180 (0.3%) were RTRs. RTRs were younger (median, 71 years vs 74.5 years; P < .001), with higher incidence of hypertension (92% vs 84%; P = .004) and diabetes (29% vs 21%; P = .005). RTRs had higher median preoperative serum creatinine (1.3 mg/dL vs 1.0 mg/dL; P < .001) and lower estimated glomerular filtration rate (51.6 mL/min vs 69.4 mL/min; P < .001). There was no difference in the abdominal aortic aneurysm diameter and incidence of concurrent iliac aneurysms. Procedurally, RTRs were more likely to undergo general anesthesia with lower amount of contrast used (median, 68.6 mL vs 94.8 ml; P < .001) and higher crystalloid infusion (median, 1700 mL vs 1500 mL; P = .039), but no difference was observed in the incidence of open conversion, endoleak, operative time, and blood loss. Postoperatively, RTRs experienced a higher rate of AKI (9.4% vs 2.7%; P < .001), but the need for new RRT was similar (1.1% vs 0.4%; P = .15). There was no difference in the rates of postoperative mortality, aortic-related reintervention, and major adverse cardiac events. After adjustment for potential confounders, RTRs remained associated with increased odds of postoperative AKI (odds ratio, 3.33; 95% confidence interval, 1.93-5.76; P < .001) but had no association with other postoperative complications. A subgroup analysis identified that diabetes (odds ratio, 4.21; 95% confidence interval, 1.17-15.14; P = .02) is associated with increased odds of postoperative AKI among RTRs. At 5 years, the overall survival rates were similar (83.4% vs 80%; log-rank P = .235). CONCLUSIONS: Among patients undergoing elective infrarenal EVAR, RTRs were independently associated with increased odds of postoperative AKI, without increased postoperative renal failure requiring RRT, mortality, endoleak, aortic-related reintervention, or major adverse cardiac events. Furthermore, 5-year survival was similar. As such, while EVAR may confer comparable benefits and technical success perioperatively, RTRs should have aggressive and maximally optimized renal protection to mitigate the risk of postoperative AKI.

Intraoperative Infusion of Dextran Confers No Additional Benefit after Carotid Endarterectomy but Is Associated with Increased Perioperative Major Adverse Cardiac Events.

BACKGROUND: Intraoperative dextran infusion has been associated with reduction of an embolic risk in patients undergoing carotid endarterectomy (CEA). Nonetheless, dextran has been associated with adverse reactions, including anaphylaxis, hemorrhage, cardiac, and renal complications. Herein, we aimed to compare the perioperative outcomes of CEA stratified by the use of intraoperative dextran infusion using a large multiinstitutional dataset. METHODS: Patients undergoing CEA between 2008 and 2022 from the Vascular Quality Initiative database were reviewed. Patients were categorized by use of intraoperative dextran infusion, and demographics, procedural data, and in-hospital outcomes were compared. Logistic regression analysis was utilized to adjust for differences in patients while assessing the association between postoperative outcomes and intraoperative infusion of dextran. RESULTS: Of 140,893 patients undergoing CEA, 9,935 (7.1%) patients had intraoperative dextran infusion. Patients with intraoperative dextran infusion were older with lower rates of symptomatic stenosis (24.7% vs. 29.3%; P < 0.001) and preoperative use of antiplatelets, anticoagulants and statins. Additionally, they were more likely to have severe carotid stenosis (>80%; 49% vs. 45%; P < 0.001) and undergo CEA under general anesthesia (96.4% vs. 92.3%; P < 0.001), with a more frequent use of shunt (64.4% vs. 49.5%; P < 0.001). After adjustment, multivariable analysis showed that intraoperative dextran infusion was associated with higher odds of in-hospital major adverse cardiac events (MACE), including myocardial infarction [MI] (odds ratio [OR], 1.76, 95% confidence interval [CI]: 1.34-2.3, P < 0.001), congestive heart failure [CHF] (OR, 2.15, 95% CI: 1.67-2.77, P = 0.001), and hemodynamic instability requiring vasoactive agents (OR, 1.08, 95% CI: 1.03-1.13, P = 0.001). However, it was not associated with decreased odds of stroke (OR, 0.92, 95% CI: 0.74-1.16, P = 0.489) or death (OR, 0.88, 95% CI: 0.58-1.35, P = 0.554). These trends persisted even when stratified by symptomatic status and degree of stenosis. CONCLUSIONS: Intraoperative infusion of dextran was associated with increased odds of MACE, including MI, CHF, and persistent hemodynamic instability, without decreasing the risk of stroke perioperatively. Given these results, judicious use of dextran in patients undergoing CEA is recommended. Furthermore, careful perioperative cardiac management is warranted in select patients receiving intraoperative dextran during CEA.

A trimodular family 16 glycoside hydrolase from the cellulosome of Ruminococcus flavefaciens displays highly specific licheninase (EC 3.2.1.73) activity.

Cellulosomes are highly complex cell-bound multi-enzymatic nanomachines used by anaerobes to break down plant carbohydrates. The genome sequence of Ruminococcus flavefaciens revealed a remarkably diverse cellulosome composed of more than 200 cellulosomal enzymes. Here we provide a detailed biochemical characterization of a highly elaborate R. flavefaciens cellulosomal enzyme containing an N-terminal dockerin module, which anchors the enzyme into the multi-enzyme complex through binding of cohesins located in non-catalytic cell-bound scaffoldins, and three tandemly repeated family 16 glycoside hydrolase (GH16) catalytic domains. The DNA sequence encoding the three homologous catalytic domains was cloned and hyper-expressed in Escherichia coli BL21 (DE3) cells. SDS-PAGE analysis of purified His6 tag containing RfGH16_21 showed a single soluble protein of molecular size ~89 kDa, which was in agreement with the theoretical size, 89.3 kDa. The enzyme RfGH16_21 exhibited activity over a wide pH range (pH 5.0-8.0) and a broad temperature range (50-70 °C), displaying maximum activity at an optimum pH of 7.0 and optimum temperature of 55 °C. Substrate specificity analysis of RfGH16_21 revealed maximum activity against barley ß-d-glucan (257 U mg-1) followed by lichenan (247 U mg-1), but did not show significant activity towards other tested polysaccharides, suggesting that it is specifically a ß-1,3-1,4-endoglucanase. TLC analysis revealed that RfGH16_21 hydrolyses barley ß-d-glucan to cellotriose, cellotetraose and a higher degree of polymerization of gluco-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a fairly high, active and thermostable bacterial endo-glucanase which may find considerable biotechnological potentials.

Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase.

The integrity of our DNA is challenged daily by a variety of chemicals that cause DNA base alkylation. DNA alkylation repair is an essential cellular defence mechanism to prevent the cytotoxicity or mutagenesis from DNA alkylating chemicals. Human oxidative demethylase ALKBH3 is a central component of alkylation repair, especially from single-stranded DNA. However, the molecular mechanism of ALKBH3-mediated damage recognition and repair is less understood. We report that ALKBH3 has a direct protein-protein interaction with human RAD51 paralogue RAD51C. We also provide evidence that RAD51C-ALKBH3 interaction stimulates ALKBH3-mediated repair of methyl-adduct located within 3'-tailed DNA, which serves as a substrate for the RAD51 recombinase. We further show that the lack of RAD51C-ALKBH3 interaction affects ALKBH3 function in vitro and in vivo. Our data provide a molecular mechanism underlying upstream events of alkyl adduct recognition and repair by ALKBH3.

Separation and characterization of cellulose from sugarcane tops and its saccharification by recombinant cellulolytic enzymes.

In the present study, the cellulose from sugarcane tops (SCT) was separated and characterized for its purity. Approximately, 85% (w/w) of total cellulose present in raw SCT was recovered by using alkaline method. The monosaccharide analysis of SCT cellulose by HPLC showed 91% D-glucose, 7.5% D-xylose and 1.5% D-arabinose residues. Surface morphology study of dried cellulosic fibers by FESEM exhibited the fibrous structure. The FTIR analysis of separated cellulose displayed the peaks corresponding to the peaks obtained from commercial cellulose, confirming its purity. The crystallinity index (CrI) of separated cellulose increased to 49% after delignification and xylan extraction from 36% of raw SCT. The typical TGA curve of separated SCT cellulose showed decomposition and mass reduction at 327 °C resulting in single decomposition peak in TGA analysis, confirming its purity. CHNS analysis supported the purity of separated cellulose by confirming absence of nitrogen and sulfur. The separated cellulose was hydrolyzed by recombinant endo-ß-1,4-glucanase (CtCel8A), cellobiohydrolase (CtCBH5A) from Clostridium themocellum and ß-1,4-glucosidase (HtBgl) from Hungateiclostridium thermocellum at pH 5.8, 50 °C for 24 h, resulting in the production of 188 mg/g of total reducing sugar (TRS). The separated cellulose from SCT can be utilized as an alternative substrate for commercialization and for bioethanol production.

Role of glycine 256 residue in improving the catalytic efficiency of mutant endoglucanase of family 5 glycoside hydrolase from Bacillus amyloliquefaciens SS35.

Wild-type, BaGH5-WT and mutant, BaGH5-UV2 (aspartate residue mutated to glycine), endoglucanases belonging to glycoside hydrolase family 5 (GH5), from wild-type, and UV2 mutant strain of Bacillus amyloliquefaciens SS35, respectively, were earlier cloned in pHTP0 cloning vector. In this study, genes encoding BaGH5-WT or BaGH5-UV2 were cloned into pET28a(+) expression-vector and expressed in Escherichia coli BL-21(DE3)pLysS cells. BaGH5-UV2 showed 10-fold (43.6 U/mg) higher specific activity against carboxymethylcellulose sodium salt (CMC-Na), higher optimal temperature by 10°C at 65°C, and 22-fold higher catalytic efficiency against CMC-Na, than BaGH5-WT. BaGH5-UV2 showed stability in wider acidic pH range (5.0-7.0) unlike BaGH5-WT in narrow basic pH range (7.0-7.5). BaGH5-UV2 displayed a mutation, Asp256Gly in L11 loop, connecting ß6 -sheet with α6 -helix, near active site toward the domain surface of (α/ß)8 -TIM barrel fold. Molecular dynamics simulation studies showed more stable structure, accessibility of substrate for a catalytic site, and increased flexibility of loop L11 of BaGH5-UV2 than the wild type, suggesting enhanced catalysis by BaGH5-UV2. Molecular docking analysis displayed enhanced hydrogen bond interactions of cello-oligosaccharides with BaGH5-UV2, unlike BaGH5-WT. Thus, Gly256 residue of loop L11 plays an important role in enhancing catalytic efficiency, and pH stability of GH5 endoglucanase. Therefore, these results help in protein engineering of GH5 endoglucanase for improved biochemical properties.

Assessment of combination of pretreatment of Sorghum durra stalk and production of chimeric enzyme (ß-glucosidase and endo ß-1,4 glucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) for saccharification to produce bioethanol.

Optimization of pretreatment and saccharification of Sorghum durra stalk (Sds) was carried out. The chimeric enzyme (CtGH1-L1-CtGH5-F194A) having ß-glucosidase (CtGH1) and endo ß-1,4 glucanase activity (CtGH5-F194A) and cellobiohydrolase (CtCBH5A) from Clostridium thermocellum were used for saccharification. Chimeric enzyme will save production cost of two enzymes, individually. Stage 2 pretreatment by 1% (w/v) NaOH assisted autoclaving + 1.5% (v/v) dilute H2SO4 assisted oven heating gave lower total sugar yield (366.6 mg/g of pretreated Sds) and total glucose yield (195 mg/g of pretreated Sds) in pretreated hydrolysate with highest crystallinity index 55.6% than the other stage 2 pretreatments. Optimized parameters for saccharification of above stage 2 pretreated biomass were 3% (w/v) biomass concentration, enzyme (chimera: cellobiohydrolase) ratio, 2:3 (U/g) of biomass, total enzyme loading (350 U/g of pretreated biomass), 24 h and 30 °C. Best stage 2 pretreated Sds under optimized enzyme saccharification conditions gave maximum total reducing sugar yield 417 mg/g and glucose yield 285 mg/g pretreated biomass in hydrolysate. Best stage 2 pretreated Sds showed significantly higher cellulose, 71.3% and lower lignin, 2.0% and hemicellulose, 12.2% (w/w) content suggesting the effectiveness of method. This hydrolysate upon SHF using Saccharomyces cerevisiae under unoptimized conditions produced ethanol yield, 0.12 g/g of glucose. Abbreviation: Ct-Clostridium thermocellum, Sds-Sorghum durra stalk, TRS-Total reducing sugar, HPLC-High performance liquid chromatography, RI-Refractive index, ADL-acid insoluble lignin, GYE-Glucose yeast extract, MGYP-Malt glucose yeast extract peptone, SHF-separate hydrolysis and fermentation, OD-Optical density, PVDF-Poly vinylidene fluoride, TS-total sugar, FESEM-Field emission scanning electron microscopy, XRD-X-ray diffraction, FTIR-Fourier transform infra-red spectroscopy and CrI-Crystallinity index.

Molecular organization and protein stability of the Clostridium thermocellum glucuronoxylan endo-ß-1,4-xylanase of family 30 glycoside hydrolase in solution.

Glucuronoxylan-ß-1,4-xylanohydrolase from Clostridium thermocellum (CtXynGH30) hydrolyzes ß-1,4-xylosidic linkages in 4-O-Methyl-D-glucuronoxylan. CtXynGH30 comprises an N-terminal catalytic domain, CtXyn30A, joined by a typical linker sequence to a family 6 carbohydrate-binding module, termed CtCBM6. ITC, mass spectrometric and enzyme activity analyses of CtXyn30A:CtCBM6 (1:1 M ratio), CtXyn30A and CtXynGH30 showed that the linker peptide plays a key role in connecting and orienting CtXyn30A and CtCBM6 modules resulting in the enhanced activity of CtXynGH30. To visualize the disposition of the two protein domains of CtXynGH30, SAXS analysis revealed that CtXynGH30 is monomeric and has a boot-shaped molecular envelope in solution with a Dmax of 18 nm and Rg of 3.6 nm. Kratky plot displayed the protein in a fully folded and flexible state. The ab initio derived dummy atom model of CtXynGH30 superposed well with the modelled structure.

Prebiotic Chondroitin Sulfate Disaccharide Isolated from Chicken Keel Bone Exhibiting Anticancer Potential Against Human Colon Cancer Cells.

Chondroitin sulfate (CS)-Keel disaccharide (CSD) was produced by chondroitin AC lyase (PsPL8A) degradation of food grade CS-Keel polysaccharide isolated from chicken keel cartilage. PsPL8A showed specific activity, 340 ± 5.2 U mg-1 with CS-Keel polysaccharide. TLC showed CSD as the major product. CSD was purified by gel filtration and MS/MS, confirms it as C4S disaccharide. Structural characterization by FTIR and NMR showed presence of N-acetylgalactosamine and glucuronic acid. CSD displayed resistance to gastric juice with 23.7% hydrolysis at pH 1.0. CSD showed prebiotic score of 0.57 for L. acidophilus and 0.58 for B. infantis and produce SCFA products. MTT and morphological analysis confirmed, CSD (0.5 mg mL-1) does not decrease viability of mouse fibroblast (L929), but showed anti-proliferative potential against human colon cancer (HT-29) cell lines (80% inhibition). CSD treated HT-29 cells exhibit nuclear fragmentation and apoptosis. Prebiotic and anticancer potential of CSD can be utilized for functional food preparation and prevention of gastrointestinal disorders.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted ß-glucans, ß-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize ß-glucans and ß-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I.

Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase (CtRGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl-CBM35. In this study we show that Rgl-CBM35 displays binding with ß-d-glucuronic acid (ß-D-GlcpA), Δ4,5-anhydro-d-galactopyranosyluronic acid (Δ4,5-GalpA), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl-CBM35 contains a conserved ligand binding site in the loops known for binding ß-D-GlcpA and Δ4,5-GalpA moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding ß-D-GlcpA, Δ4,5-GalpA, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl-CBM35 showed no affinity towards ß-D-GlcpA and Δ4,5-GalpA underscoring Ca2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl-CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl-CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites.

SAXS and homology modelling based structure characterization of pectin methylesterase a family 8 carbohydrate esterase from Clostridium thermocellum ATCC 27405.

Pectin methylesterase (CtPME) from Clostridium thermocellum of family 8 carbohydrate esterase (CE8) belongs to pectin methylesterase super family (E.C.3.1.1.11). BLAST analysis of CtPME showed 38% sequence identity with PME from Erwinia chrysanthemi. Multiple sequence alignment of CtPME with other known structures of pectin methylesterase revealed the conserved and semi-conserved amino acid residues. Homology modelling of CtPME structure revealed a characteristic right handed parallel ß-helices. The energy of modelled structure was minimized by using YASARA software. The Ramachandran plot of CtPME shows 83.7% residues in non-glycine and non-proline residues in most-favorable region, 13.8% in additional allowed region and 1.4% in generously allowed region, indicating that CtPME has a stable conformation. The secondary structure of CtPME predicted using PSI-Pred software and confirmed by the circular dichroism (CD) showed α-helices (3.1%), ß-sheets (40.1%) and random coils (56.9%). Small Angle X-ray Scattering (SAXS) analysis demonstrated the overall shape and structural characterization of CtPME in solution form. Guinier analysis gave the radius of gyration (Rg) 2.28 nm for globular shape and 0.74 nm for rod shape. Kratky plot gave the indication that protein is fully folded in solution. The ab initio derived dummy atom model of CtPME superposed well on modelled CtPME structure.

Comparative analysis of pretreatment methods on sorghum (Sorghum durra) stalk agrowaste for holocellulose content.

This study compares different types of pretreatment methods, such as thermal pretreatment at 120 °C, autoclaving, microwaving and ultrasonication in the presence of water, dilute acid (1% H2SO4) or dilute alkali (1% NaOH) on Sorghum stalk with respect to the holocellulose and Acid Detergent/Insoluble Lignin content. Among all the methods, pretreatment with 1% NaOH along with autoclaving at 121 °C and 15 psi for 30 min was the most effective method for Sorghum stalk. Fourier Transform Infra-Red spectroscopy analysis of this pretreated biomass showed the removal of lignin and Field Emission Scanning Electron Microscope analysis displayed enhanced surface roughness. The enzymatic hydrolysis of raw and best pretreated Sorghum stalk using recombinant endo-ß-1,4-glucanase (CtCel8A) and ß-1,4-glucosidase (CtBgl1A) both from Clostridium thermocellum gave glucose yields, 22.4 mg/g raw biomass and 34 mg/g pretreated biomass, respectively, resulting in 1.5-fold increase of glucose yield after the pretreatment.

Rye bran as fermentation matrix boosts in situ dextran production by Weissella confusa compared to wheat bran.

The consumption of fiber-rich foods such as cereal bran is highly recommended due to its beneficial health effects. Pre-fermentation of bran with lactic acid bacteria can be used to improve the otherwise impaired flavor and textural qualities of bran-rich products. These positive effects are attributed to enzymatic modification of bran components and the production of functional metabolites like organic acids and exopolysaccharides such as dextrans. The aim of this study was to investigate dextran production in wheat and rye bran by fermentation with two Weissella confusa strains. Bran raw materials were analyzed for their chemical compositions and mineral content. Microbial growth and acidification kinetics were determined from the fermentations. Both strains produced more dextran in rye bran in which the fermentation-induced acidification was slower and the acidification lag phase longer than in wheat bran. Higher dextran production in rye bran is expected to be due to the longer period of optimal pH for dextran synthesis during fermentation. The starch content of wheat bran was higher, which may promote isomaltooligosaccharide formation at the expense of dextran production. W. confusa Cab3 produced slightly higher amounts of dextran than W. confusa VTT E-90392 in all raw materials. Fermentation with W. confusa Cab3 also resulted in lower residual fructose content which has technological relevance. The results indicate that wheat and particularly rye bran are promising matrices for producing technologically significant amounts of dextran, which facilitates the use of nutritionally valuable raw bran in food applications.

Enzyme-resistant isomalto-oligosaccharides produced from Leuconostoc mesenteroides NRRL B-1426 dextran hydrolysis for functional food application.

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1426 was produced and purified using polyethylene glycol fractionation. In our earlier study, it was reported that L. mesenteroides dextransucrase synthesizes a high-molecular mass dextran (>2 × 10(6)  Da) with ∼85.5% α-(1→6) linear and ∼14.5% α-(1→3) branched linkages. Isomalto-oligosaccharides (IMOs) were synthesized through depolymerization of dextran by the action of dextranase. The degree of polymerization of IMOs was 2-10 as confirmed by mass spectrometry. The nuclear magnetic resonance spectroscopic analysis revealed the presence of α-(1→3) linkages in the synthesized IMOs. The IMOs were resistant to dextranase, α-glucosidase, and α-amylase, and therefore can have potential application as food additives in the functional foods.

Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis.

The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2 kb coding for 1,402 amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5 U/mg which after purification by PEG-400 fractionation gave 6.1 U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180 kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50 mM sodium acetate buffer. WcCab3-rDSR gave Km of 6.2 mM and Vm of 6.3 µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (ß/α)8 triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A.

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ~14 kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca(2+) ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% ß-strands supporting the predicted structure. CtCBM6 displays a jelly roll ß-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting ß4-ß5 and ß8-ß9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of ß-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

Subcutaneous emphysema: Unique presentation of a foreign body in the airway.

Foreign body airway (FBA) is a common problem among the children. Variable presentation makes it difficult to diagnose a case of FBA, particularly, when no definite history of aspiration is available. Subcutaneous emphysema (SCE) and pneumomediastinum are rare presentations. We report a case of FBA who presented with SCE without any history of aspiration. A 3-year-old female child was admitted with respiratory distress, fever and SCE over the right side of chest, neck and face. Initially, she was diagnosed as a case of pneumonitis with barotrauma. X-ray of the chest revealed SCE with pneumomediastinum without pneumothorax. Diagnostic bronchoscopy with rigid ventilating bronchoscope was done under general anesthesia. A plastic foreign body with sharp projections embedded in the mucosa was detected and retrieved from right main bronchus. Postoperatively SCE regressed gradually.

Potential probiotic attributes and antagonistic activity of an indigenous isolate Lactobacillus plantarum DM5 from an ethnic fermented beverage "Marcha" of north eastern Himalayas.

A novel isolate DM5 identified as Lactobacillus plantarum displayed in vitro probiotic properties as well as antimicrobial activity. It showed adequate level of survival to the harsh conditions of the gastrointestinal tract and survived low acidic pH 2.5 for 5 h. Artificial gastric juice and intestinal fluidic environment decreased the initial viable cell population of isolate DM5 only by 7% and 13%, respectively, while lysozyme (200 µg/ml) and bile salt (0.5%) enhanced its growth. It was found to deconjugate taurodeoxycholic acid, indicating its potential to reduce hypercholesterolemia. Isolate DM5 demonstrated cell surface hydrophobicity of 53% and autoaggregation of 54% which are the prerequisite for adhesion to epithelial cells and colonization to host. Bacteriocin activity of isolate was found to be 6400 AU/ml as it inhibited the growth of food borne pathogens Escherichia coli, Staphylococcus aureus, and Alcaligenes faecalis. The bactericidal action of bacteriocin from isolate was analyzed by flow cytometry, rendering its use as prospective probiotic and starter culture in food industry.