Sodium Citrate Buffer Research Articles

Enhancement of bio-hydrogen yield and pH stability in photo fermentation process using dark fermentation effluent as succedaneum

The photo fermentation hydrogen yield from dark fermentation effluents (DFEs) can be promoted by adding corn straw enzymatic hydrolysate adjusts the nutritional composition of DFEs. As compared with the control group (without enzymatic hydrolysate addition), the effect of adding enzymatic hydrolysate make H2 yield increase from 312.54 to 1287.06 mL H2/g TOC, and maximum hydrogen production rate increase 2.14 to 10.23 mL/h. On the other hand, buffer reagents remained in DFEs make which can replace part sodium citrate buffer to maintain pH stability in synchronized saccharification and photosynthetic fermentation process with corn straw as substrate, the best result was observed at the ration of 1:2 (33 mL DFEs, 67 mL sodium citrate buffer) with the hydrogen yield of 436.30 ± 10 mL, and which can cut down the GHG in the life cycle of hydrogen production.

Sustainable use of the spent mushroom substrate of Pleurotus florida for production of lignocellulolytic enzymes.

Spent mushroom substrate (SMS), a major byproduct of the mushroom industry, is a lignocellulosic biomass, which contains approximately 57-74.3% of holocellulose fraction. This study was aimed at utilizing SMS of Pleurotus florida for recovery of lignocellulolytic enzymes and sugars and also as a substrate for production of cellulolytic enzymes using different isolates of Trichoderma and Aspergillus under solid-state fermentation (SSF). SMS of P. florida extracts contained significant amounts of laccase (3,015.8 ± 29.5 U/g SMS) and xylanase (1,187.9 ± 12 U/g SMS) activity. Crystallinity pattern and chemical changes in SMS revealed that SMS had a lower crystallinity index (34.2%) as compared with the raw biomass (37.8%), which, in turn, helps in enhancing the accessibility of cellulolytic enzymes to holocellulose. Among the isolates, Trichoderma longibrachiatum A-01 showed maximum activity of endoglucanase (220.4 ± 5.9 U/mg), exoglucanase (78.5 ± 3.2 U/mg) and xylanase (1,550.4 ± 11.6 U/mg) while Aspergillus aculeatus C-08 showed maximum activity of cellobiase (113.9 ± 3.9 U/mg). Extraction with sodium citrate buffer (pH 4.8) showed maximum cellulolytic enzyme activity as compared with other solvents tested. Partial purification of endoglucanase, exoglucanase, xylanase, and cellobiase resulted in 56.3% (1,112.5 U/mg), 48.4% (212.5 U/mg), 44% (4,492.3 U/mg), and 62% (705.0 U/mg) yield with an increase by 5.2-, 4.5-, 4.1-, and 5.0-fold as compared with crude extract. The results reveal that SMS from P. florida could be a potential and cost-effective substrate for production of cellulolytic enzymes from T. longibrachiatum A-01 and A. aculeatus C-08.

Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody.

Aggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R2= 0.92). The model includes secondary virial coefficient, B22, as the representative parameter of colloidal stability, and aggregation temperature, Tagg, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.

Mesenchymal stem cell-derived exosomes improve diabetes mellitus-induced myocardial injury and fibrosis via inhibition of TGF-β1/Smad2 signaling pathway

The aim of this study is to investigate the effect of mesenchymal stem cell (MSC)-derived exosomes on diabetes mellitus-induced myocardial injury, and the underlying mechanism. Thirty adult male Sprague Dawley rats were randomly assigned to three groups of ten rats each: normal control group, diabetic control group and MSC exosomes group. Exosomes were isolated from MSCs through gradient ultracentrifugation. With the exception of normal control, diabetes mellitus (DM) was induced in the rats with a single intraperitoneal injection of 30 mg/kg body weight streptozotocin (STZ) in 0.1 mol/L sodium citrate buffer. Rats in MSC exosomes group were intravenously injected with MSC-derived exosomes once a week for 12 weeks. Left ventricular collagen (LVC) level was measured using acid hydrolysis method. Fatty acid transporters (FATPs) and fatty acid beta oxidase (FA-β-oxidase) were determined using enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of TGF-β and Smad2 were determined using real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting. Flow cytometric analysis and Western blotting revealed positive expression of exosomal specific marker, CD63.The level of LVC was significantly higher in diabetic control group than in normal control group, but was significantly reduced after treatment with MSC-derived exosomes (p < 0.05). The levels of FATPs and FA-β-oxidase were significantly lower in diabetic control group than in normal control group (p < 0.05). However, treatment with MSC-derived exosomes significantly increased the levels of these proteins (p < 0.05). The levels of expression of TGF-β1 and Smad2 mRNAs were significantly higher in the diabetic control group than in normal control group, but were significantly reduced after treatment with MSC-derived exosomes (p < 0.05). The expressions of TGF-β1 and Smad2 proteins were also significantly upregulated in diabetic control group, when compared with normal control group (p < 0.05). However, treatment with MSC-derived exosomes significantly down-regulated the expression of these proteins (p < 0.05). The results obtained in this study indicate that MSC-derived exosomes improve DM-induced myocardial injury and fibrosis via inhibition of TGF-β1/Smad2 signaling pathway.

Purification, characterization and application of bromelain from Ananas comosus

Bromelain is a major protease, isolated from pineapple (Ananas comosus). Bromelain is accumulated in the entire plant to a different extent and properties depending on its source. In the present study, Bromelain was extracted from all parts of pineapple using sodium citrate buffer. Bromelain was filtered, centrifuged and used for further studies. After the determination of protease activity and protein content, the Core and Pulp extract of A.comosus was chosen using gelatin as the substrate. The Bromelain was purified by precipitation and dialysis. Then SDS-PAGE was performed in order to determine the molecular size of the obtained protein. Then the effectiveness of Bromelain as an anti-browning agent, extracted and purified from pineapple Core and Pulp was determined. The study showed that Bromelain is a better anti-browning agent when compared with some of the available commercial anti-browning agents. Further, the application of Bromelain was tested by stain removal, compared to the positive control, the ability of stain-removing property for the produced enzyme was observed to be good. Immobilized Bromelain prepared using calcium alginate beads and its stability and characters were noted.

The Influence of UV Irradiation on Diabetic Mice Skin: A Vibrational FT-IR and Raman Spectroscopic Study

Forty-two (42) SKH-2 hairless male mice were divided into six groups consisting of 7 mice each. The control non diabetic (N) mice and irradiated control (IrN) mice; the diabetic mice injected with 20 mg/kg SZT (D20) and the corresponding irradiated (IrD20); the diabetic mice injected with 30 mg/kg SZT (D30) and the irradiated mice (IrD30). Diabetes was induced by given to each mouse five daily intraperitoneal IP injections of SZT (20 or 30 mg/kg) solution in sodium citrate buffer 0.1 M, pH 3.5-4.5.

Impact of ripening inhibitors on molecular transport of antimicrobial components from essential oil nanoemulsions

The objective of this study was to provide insights into the mechanisms involved in the mass transport of antimicrobial compounds from essential oil nanoemulsions to bacterial cell membranes. Origanum oil-in-water nanoemulsions were produced using spontaneous emulsification by titrating a mixture of essential oil, ripening inhibitor, and surfactant (Tween 80) into 5 mM sodium citrate buffer (pH 3.5). Stable nanoemulsions containing relatively small droplets (d < 60 nm) were produced using this low-energy method. The nature of the ripening inhibitor used in the oil phase of the nanoemulsions affected the antimicrobial activity of the nanoemulsions: corn (LCT) > medium-chain triglycerides (MCT). Differences in antimicrobial activity were attributed to the differences in the rate of transfer of hydrophobic antimicrobial constituents from the nanoemulsion to the MCT emulsion, which was used to mimic the hydrophobic region of the bacterial cell membranes. Each antimicrobial nanoemulsion was separated from the MCT emulsion by a dialysis tubing. Dialysis tubing with two different pore sizes was used, one excluding nanoemulsion droplet and micelle delivery, allowing the delivery of antimicrobial compounds only through the aqueous phase and the other by both the aqueous phase and micelles. For origanum oil nanoemulsions, the delivery of all antimicrobial agents occurred more efficiently when micelles were present.

Freeze-dry processing of three-dimensional cell constructs for bone graft materials.

Freeze-dry processing improves the operability and stability of cell-based biomaterials and facilitates sterilization for clinical application. However, there is no established freeze-drying protocol for engineered tissues. Recently, we reported that biomimetic bone tissues can be fabricated using scaffold-free three-dimensional (3D) cell constructs with potential applications as bone graft materials. The purpose of this study was to assess the influence of freeze drying on the morphology and components of 3D cell constructs. Cell constructs freeze dried in phosphate buffered saline (PBS) maintained organic and inorganic components; whereas sodium citrate buffer (SCB)-treated constructs had significantly lower amounts of calcium and bone-related proteins. Alkaline phosphatase (ALP) activity in cell constructs was maintained by freeze drying in 10% sucrose-containing PBS, whereas cell constructs treated with PBS without sucrose or with sucrose-containing SCB showed significant reductions of ALP activity. In this study, we found that sucrose-containing phosphate buffer was suitable for freeze drying to maintain minerals and protein functions within 3D cell constructs, whereas citrate buffer was inappropriate. The insights gained by this study may facilitate the development of novel cell-based biomaterials fabricated by tissue engineering approaches and bone graft biomaterials.

Utilisation of protein fractions from porcine spleen as technofunctional ingredients in emulsified cooked meat sausages

SummarySpleen is a low commercial value meat by‐product from industrial slaughterhouses that is mostly underutilised. In this study, porcine spleens were processed to obtain two protein fractions (i.e. soluble and insoluble extracts in sodium citrate buffer 0.1 m pH 5) and the potential of using them to replace functional ingredients in cooked sausages was assessed. Specifically, a pasteurised insoluble protein extract was used as soy protein replacer in mortadella‐type sausages, whereas spray‐dried soluble proteins were used instead of sodium caseinate in Frankfurt‐type sausages. Both splenic extracts performed well as functional ingredients since no differences in proximate composition, emulsion stability, water‐holding capacity or texture were found. The only differences were found in the colour of the sausages containing the soluble protein extract, because of the haem and myoglobin content in this ingredient. The results showed that spleens could be a suitable source of non‐allergenic functional proteins for meat products.

Exogenous fibrolytic enzymes and recombinant bacterial expansins synergistically improve hydrolysis and in vitro digestibility of bermudagrass haylage

Four experiments were conducted to examine the effects of a recombinant bacterial expansin-like protein (BsEXLX1) from Bacillus subtilis and a commercial exogenous fibrolytic enzyme (EFE) preparation for ruminants on hydrolysis of pure substrates (cellulose and xylan) and in vitro digestibility of bermudagrass haylage (BMH). Recombinant Escherichia coli BL21 strain was used to express BsEXLX1; the protein was purified using an affinity column. In experiment 1, carboxymethylcellulose, Whatman #1 filter paper (General Electric, Boston, MA) and oat-spelt xylan substrates were subjected to 4 treatments (1) sodium citrate buffer (control), (2) BsEXLX1 (162 µg/g of substrate), (3) EFE (2.3 mg/g of substrate), and (4) EFE + BsELX1 in 3 independent runs. Samples were incubated at optimal conditions for both additives (pH 5 and 50°C) or at ruminal (pH 6 and 39°C) or ambient (pH 6 and 25°C) conditions for 24 h and sugar release was measured. In experiment 2, digestibility in vitro of BMH was examined after treatment with the following: (1) control (buffer only), (2) BsEXLX1 (162 µg/g of dry matter), (3) EFE (2.2 mg/g of dry matter), and (4) EFE + BsEXLX1 in 3 independent runs at 39°C for 24 h. Experiment 3 examined effects of EFE and BsEXLX1 on simulated preingestive hydrolysis and profile of released sugars from BMH after samples were suspended in deionized water with sodium azide at 25°C for 24 h in 2 independent runs. In experiment 4, the sequence of the BsEXLX1 purified protein was compared with 447 ruminal bacterial genomes to identify similar proteins from the rumen. In experiment 1, compared with EFE alone, EFE and BsEXLX1 synergistically increased sugar release from carboxymethylcellulose and Whatman #1 filter paper under all simulated conditions; however, hydrolysis of xylan was not improved. In experiment 2, compared with EFE alone, treatment with EFE and BsEXLX1 increased neutral detergent fiber and acid detergent fiber digestibility of bermudagrass haylage (by 5.5 and 15%, respectively) and total volatile fatty acid concentrations, and decreased acetate-propionate ratio. In experiment 3, compared with EFE alone. The EFE and BsEXLX1 synergistically reduced concentrations of neutral detergent fiber and acid detergent fiber and increased release of sugars by 9.3%, particularly cellobiose (72.5%). In experiment 4, a similar sequence to that of BsEXLX1 was identified in Bacillus licheniformis, and similar hypothetical protein sequences were identified in Ruminococcus flavefaciens strains along with different protein structures in E. xylanophilum and Lachnospiraceae. This study showed that an expansin-like protein synergistically increased the hydrolysis of pure cellulose substrates and the hydrolysis and digestibility in vitro of BMH.

Evaluation of Enrofloxacin for Use in Cryopreservation of Zebu Bull (Bos indicus) Semen.

Aim: The present study was designed to evaluate enrofloxacin (EN), a fluoroquinolone broad-spectrum antibacterial drug for use in cryopreservation of Zebu bull semen. Materials and Methods: Semen was collected from three Zebu bulls of the Sahiwal breed (Bos indicus) for 3 weeks (replicates) for each experiment. Experiment I: semen samples were cultured for isolation of bacteria and in vitro sensitivity tests against streptomycin, penicillin, and EN. Proteus spp., Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were isolated. P. aeruginosa was found to be resistant to streptomycin and penicillin (SP), while Proteus spp. was resistant to streptomycin. EN effectively inhibited the growth of all four bacteria in an in vitro sensitivity test. Experiment II: in vitro dose toxicity of EN (0-2000 μg/mL) for bull sperm was assessed in 2.9% sodium citrate buffer. EN up to a concentration of 1200 μg/mL was found to be safe for motility and viability of bull spermatozoa. Experiment III: semen was cryopreserved in a tris-citric acid extender containing either SP or EN (400 μg/mL) or without antibiotics as the control. Results: Post-thaw sperm motility was found to be higher in the extender containing EN and SP compared to control. Nevertheless, sperm viability was recorded higher (p < 0.05) with EN compared to SP and control. Sperm chromatin integrity did not differ (p > 0.05) in EN, SP, and controls. The total aerobic bacterial count was recorded as zero in the extender containing EN compared to SP and control. The fertility rate was higher (p < 0.05) with 400 μg/mL EN (59%) compared to SP (44%). Conclusions: It is concluded that EN is capable of controlling the bacterial load in frozen Zebu bull semen, resulting in a higher fertility rate under field conditions.

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

Next-Generation Sequencing Identifies the Keratinocyte-Specific miRNA Signature of Psoriasis

Citrate-assisted efficient local delivery of naked oligonucleotide into live mouse brain cells.

ObjectivesSynthetic oligonucleotides have shown promise in brain imaging. However, delivery of oligonucleotides into live brain cells remains challenging. In this study, we aim to develop a facile yet efficient strategy for local delivery of oligodeoxynucleotide (ODN) to neural cells in live adult mouse brain.Materials and methodsA fluorescence‐labelled ODN was diluted with sodium citrate buffer (100 mmol/L, pH = 3). One microlitre of the mixture was injected into a live adult mouse brain. Six hours later, we sacrificed the mouse and prepared brain slices for microscopic imaging.ResultsWe find that the use of sodium citrate buffer in the one‐shot local delivery can improve the diffusion and cell entry efficiency of the unmodified ODN for dozens of times. Only 1 pmol ODN leads to hundreds of positively transferred brain cells. We reason that this promotion is due to the local acidic condition created by the citrate buffer, which leads to the protonation of the ODN and some membrane proteins, thus reduces the Coulomb repulsion between the ODN and the cell membrane. Based on this strategy, we demonstrate fluorescent microscopic imaging of brain cells in different brain regions including striatum, cortex, hippocampus and midbrain.ConclusionsThe citrate buffer can be used as an adjuvant for facile and effective local injection delivery of ODNs, which may provide a new tool for brain imaging.

Treatment of Diabetic Condition in Streptozotocin Induced Diabetic Wister Rats Using Food Blends Such as Unripe Plantain, Soybean and Ginger

Aims: To investigate the effect of food blends (plantain, soybean and ginger) on the blood glucose, lipid profile and haematological indices on streptozotocin induced diabetic rats.&#x0D; Methodology: A total of 35 rats of mean body weight 219.07 g separated into7 groups (5 per group) where induced by a single intraperitoneal (I.P) injection of streptozotocin (0.1 g dissolved in 5 ml of freshly prepared sodium citrate buffer 0.1 M, pH 4.5) at a dose of 40 mg/kg body weight after fasting for 12 hours and fed with flours/blends. The flours were produced from plant materials for different treatments/blends (blend A=100% unripe plantain, B=80% unripe plantain, 14% soybean, 6% ginger, C=70% unripe plantain, 26% soybean, 4% ginger, D= 60% unripe plantain, 38% soybean, 2% ginger, E= 50% unripe plantain, 50% soybean) and the phytochemicals and minerals content were determined. Blood glucose was determined at 5 days interval for 25 days. Diabetes was confirmed in rats with blood glucose concentrations &gt;200 mg/dl. After 25 days rats were anaesthetized with chloroform vapour and blood samples collected by cardiac puncture for haematology and lipid profile determination.&#x0D; Results: The results showed that unripe plantain, soya beans and ginger in adequate proportion(C=70% unripe plantain, 26% soybean, 4% ginger or D= 60% unripe plantain, 38% soybean, 2% ginger) could help to reduce blood glucose, improve haematological parameters and lipid profile. Significant reduction was observed in the blood glucose level of rats fed blends C and D from 286 to 85 mg/dl and 307 to 90 mg/dl respectively at the end of experiment. These results also demonstrated that the inclusion of ginger at 6% causes rise in blood glucose level. Total cholesterol (TC) increased in all the blends. However, the lowest concentration of TC was observed in blends C and D. The highest packed cell volume (60%) and Haemoglobin (20 g/dl) level observed in rats fed blend C was significantly higher than the normal control fed conventional feeds. The increase in packed cell volume (PCV) (50%) and Hb (17 g/dl) in diabetic rats demonstrated that the formulated blend C was able to raise PCV and Hb above 50% and 17 g/dl (Normal control NC) respectively. Significant increase (P&lt;0.05) in low density lipoprotein cholesterol (LDLc) was also observed in all the blends with blend C having the least (4.0 mg/dl) close to NC (2.0 mg/dl).&#x0D; Conclusion: From the results it is evident that blend C will manage and improve the health status of diabetic patients.

Fenofibrate improves vascular endothelial function in diabetic mice

Microvascular and macrovascular complications are major causes of disability and death in diabetic patients. High levels of blood glucose sabotage the integrity of blood vessels and induce endothelial dysfunction. Fenofibrate is an agonist of peroxisome proliferator-activated receptor α and can reduce the incidence of cardiovascular events in diabetic patients. This study tested the hypothesis that fenofibrate could ameliorate endothelium-dependent vasodilation in diabetic mice and relieve high glucose-induced endothelial dysfunction via activating endothelial nitric oxide synthase (eNOS) and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A streptozotocin (STZ)-induced diabetic model was established by intraperitoneal injection of STZ (dissolved in sodium citrate buffer) at a dose of 60 mg/kg for 5 consecutive days. Mice were administered fenofibrate (100 mg/kg/d, i.g.) for 14 days. The endothelial function of extracted mouse aortae was examined by evaluating acetylcholine induced endothelium-dependent relaxation combined with phenylephrine-induced vasoconstriction and sodium nitroprusside-induced endothelium-independent relaxation. Superoxide onion (O2−) was determined using dihydroethidium staining of aortae. Functions of mouse aortic endothelial cells (MAECs) were assessed, and expression levels of eNOS and AMPK were determined by Western blotting. Fenofibrate ameliorated the impaired endothelium-dependent relaxation in diabetic mice and decreased the level of intracellular O2- in diabetic mouse aortae. In-vitro, fenofibrate treatment improved the impaired function of MAECs, increased nitric oxide production, and decreased the O2- level, as well as activated eNOS and AMPK phosphorylation in cultured MAECs by high glucose. Fenofibrate could ameliorate endothelium-dependent vasodilation in diabetic mice and relieve high glucose-induced endothelial dysfunction, which was possibly related to the activation of eNOS and AMPK phosphorylation.

Rheological behavior of soluble dietary fiber fractions isolated from artichoke residues

Cynara cardunculus var. scolymus agroindustrial by-products (stems, outer bracts, leaves) constitute 80% of the plant. These by-products may be recovered for the extraction of food additives and nutraceuticals. In this research, the rheological behavior of soluble dietary fiber fractions isolated from bracts (B) and stems (S) of artichoke was studied. Fractions were isolated by means of a heat treatment followed by processing with sodium citrate buffer (S1, B1) or buffer/protease (S2, B2) or hemicellulase (S3, B3) or both enzymes (S4, B4). They were composed, mainly, by pectin (degree of methylation, DM ≤ 53%) and inulin. When dissolved in water (2.00% w/v) with Ca2+ (40 mg/g pectin), fractions were sensitive to the ion showing shear thinning flow, with initial yield stress in the case of B2, B3 or S3. Citrate buffer extracted fractions (B1, S1) showed the highest viscosities, whereas extraction with buffer and both enzymes (B4, S4) produced the least viscous fiber fractions. At rest assays revealed the formation of gel-like networks that were stronger, in general, in the presence of higher pectin and inulin content, homogalacturonan proportion, and low methylation degree. Leftovers of artichoke can be used to extract soluble dietary fiber fractions that might be utilized as natural thickeners and gelling agents in foods, pharmaceutical and cosmetics, helping to add value to raw materials and to close the bioeconomy loop in relation to this plant.

Effects of pH on steam explosion extraction of acetylated galactoglucomannan from Norway spruce

BackgroundAcetylated galactoglucomannan (AcGGM) is a complex hemicellulose found in softwoods such as Norway spruce (Picea abies). AcGGM has a large potential as a biorefinery feedstock and source of oligosaccharides for high-value industrial applications. Steam explosion is an effective method for extraction of carbohydrates from plant biomass. Increasing the reaction pH reduces the combined severity (R^{prime}_{0}) of treatment, affecting yields and properties of extracted oligosaccharides. In this study, steam explosion was used to extract oligosaccharides from Norway spruce wood chips soaked with sodium citrate and potassium phosphate buffers with pH of 4.0–7.0. Yields, monosaccharide composition of released oligosaccharides and biomass residue, their acetate content and composition of their lignin fraction were examined to determine the impact of steam explosion buffering on the extraction of softwood hemicellulose.ResultsReducing the severity of steam explosion resulted in lower yields, although the extracted oligosaccharides had a higher degree of polymerization. Higher buffering pH also resulted in a higher fraction of xylan in the extracted oligos. Oligosaccharides extracted in buffers of pH > 5.0 were deacetylated. Buffering leads to a removal of acetylations from both the extracted oligosaccharides and the hemicellulose in the residual biomass. Treatment of the residual biomass with a GH5 family mannanase from Aspergillus nidulans was not able to improve the AcGGM yields. No hydroxymethylfurfural formation, a decomposition product from hexoses, was observed in samples soaked with buffers at pH higher than 4.0.ConclusionsBuffering the steam explosion reactions proved to be an effective way to reduce the combined severity (R^{prime}_{0}) and produce a wide range of products from the same feedstock at the same physical conditions. The results highlight the impact of chemical autohydrolysis of hemicellulose by acetic acid released from the biomass in hydrothermal pretreatments. Lower combined severity results in products with a lower degree of acetylation of both the extracted oligosaccharides and residual biomass. Decrease in severity appears not to be the result of reduced acetate release, but rather a result of inhibited autohydrolysis by the released acetate. Based on the results presented, the optimal soaking pH for fine-tuning properties of extracted AcGGM is below 5.0.

Apocynin inhibited NLRP3/XIAP signalling to alleviate renal fibrotic injury in rat diabetic nephropathy

AimsIn this animal study, we tried to test the hypothesis that apocynin could play an anti-inflammation role by inhibiting NLRP3/X-linked inhibitor of apoptosis protein (XIAP) signalling and have an effect on antifibrosis in rats with diabetic nephropathy. Main methodsDiabetic nephropathy rats were induced by tail-vein injection of streptozotocin at 60 mg/kg body weight in sodium citrate buffer (0.01 M, pH 4.5) with unrestricted access to food/water for 12 weeks, and rats with blood glucose levels above 18.0 mM were considered diabetic; the damage index for glomerular mesangial cells damage index was calculated by morphological examinations; protein and mRNA changes were analysed by western blotting immunohistochemistry and real-time quantitative polymerase chain reaction; interstitial fibrosis was assessed and scored using Masson’s staining. Key findingsIn rats with diabetic nephropathy, apocynin (1) reduced renal injury and improved renal function; (2) downregulated the expression of NLRP3 in renal cortex; (3) downregulated the expression of XIAP in renal cortex; and (4) attenuated renal fibrosis. SignificanceAs an inhibitor of reactive oxygen species (ROS), apocynin could downregulate the expression of NLRP3 and XIAP, and alleviate renal fibrosis, which meant not only that ROS was one type of ligands of NLRP3, but also that ROS mechanism and NLRP3 activation might be therapeutic targets in the treatment of diabetic nephropathy in the future.

PBMC fixation and processing for Chromium single-cell RNA sequencing

BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.MethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform.ResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3× SSC were successfully implemented into 10× Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones.ConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.

Nesfatin-1 Ameliorates Testicular Function Changes in Type 2 Diabetic Rats

Nesfatin-1 Ameliorates Testicular Function Changes in Type 2 Diabetic Rats