Assessment of functional efficacy of sheep plasma protein hydrolysates and their utilization in mutton sausage.

The present study examines the bioactive potential of sheep plasma protein hydrolysates (SPPH) produced by in-vitro gastrointestinal digestion as antioxidants, antimicrobials, anti-obesity agents, and inhibitors of lipid oxidation in sausage to address the oxidative stability and shelf-life issues of mutton. The antioxidant and antimicrobial activities, indicate a positive relationship between the degree of hydrolysis and digestion duration. The study finds that SPPH has a potent inhibitory effect on pancreatic lipase and cholesterol esterase. It has higher oil holding capacity than sheep plasma protein, observed at one hour of hydrolysis time. SPPH exhibit an improved behavior in foaming properties along alkaline pH and digestion time while display lower emulsifying activity and stability with hydrolysis advancement. The SPPH act as a natural preservative in developing functional mutton sausage by inhibiting lipid-oxidation. This study showed that the recovery of SPPH can be a cost-effective and sustainable strategy for generating available ingredients for enhanced shelf-life of meat products.

Use of Moringa oleifera leaves (sole or combined with concentrate) in rabbit feeding: Effects on performance, carcass characteristics and meat quality attributes.

Sixty Chinchilla rabbits (28 days old) were divided into three equal groups (n = 20). Rabbits in MOL0 (control) were fed pellets containing 700 g cowpea hay/kg pellet as forage source, whereas rabbits in MOL700 and MOL950 were fed pellets containing 700 and 950 g moringa leaves/kg feed pellets, respectively. Average daily gain and feed conversion ratio was comparable in MOL700 and MOL0, however, it was higher in MOL950. Among the carcass traits, dressing percent was higher while, chilling loss was lower in MOL700 treatment. The Longissimus thoracis muscle of rabbits fed moringa leaves containing pellets (MOL700 and MOL950) had lower saturated fatty acid content, higher C18:3n-3 and total n-3 fatty acids along with lower thrombogenic index value. Hence, incorporating moringa leaves at 70% level is beneficial in terms of improved growth performance and functional attributes of meat than diet- containing sole moringa leaves.

Peptidoglycan binding by a pocket on the accessory NTF2-domain of Pgp2 directs helical cell shape of Campylobacter jejuni.

The helical morphology of Campylobacter jejuni, a bacterium involved in host gut colonization and pathogenesis in humans, is determined by the structure of the peptidoglycan (PG) layer. This structure is dictated by trimming of peptide stems by the LD-carboxypeptidase Pgp2 within the periplasm. The interaction interface between Pgp2 and PG to select sites for peptide trimming is unknown. We determined a 1.6 Å resolution crystal structure of Pgp2, which contains a conserved LD-carboxypeptidase domain and a previously uncharacterized domain with an NTF2-like fold (NTF2). We identified a pocket in the NTF2 domain formed by conserved residues and located ∼40 Å from the LD-carboxypeptidase active site. Expression of pgp2 in trans with substitutions of charged (Lys257, Lys307, Glu324) and hydrophobic residues (Phe242 and Tyr233) within the pocket did not restore helical morphology to a pgp2 deletion strain. Muropeptide analysis indicated a decrease of murotripeptides in the deletion strain expressing these mutants, suggesting reduced Pgp2 catalytic activity. Pgp2 but not the K307A mutant was pulled down by C. jejuni Δpgp2 PG sacculi, supporting a role for the pocket in PG binding. NMR spectroscopy was used to define the interaction interfaces of Pgp2 with several PG fragments, which bound to the active site within the LD-carboxypeptidase domain and the pocket of the NTF2 domain. We propose a model for Pgp2 binding to PG strands involving both the LD-carboxypeptidase domain and the accessory NTF2 domain to induce a helical cell shape.

Synthesis of a meso-Oxa-Diaminopimelic Acid Containing Peptidoglycan Pentapeptide and Coupling to the GlcNAc-anhydro-MurNAc Disaccharide.

The syntheses of peptidoglycan (PG)-derived peptides containing meso-diaminopimelic acid (meso-Dap) are typically quite lengthy due to the need to prepare orthogonally protected meso-Dap. In this work, the preparation of the PG pentapeptide containing the isosteric analog meso-oxa-Dap is described. The synthesis relies on the ring opening of a peptide embedded aziridine via the attack of a serine residue. The pentapeptide was attached to a GlcNAc-anhydro-MurNAc disaccharide, to produce a putative substrate for the AmpG pore protein.

Peptides Containing meso-Oxa-Diaminopimelic Acid as Substrates for the Cell-Shape-Determining Proteases Csd6 and Pgp2.

The enzymes Csd6 and Pgp2 are peptidoglycan (PG) proteases found in the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, respectively. These enzymes are involved in the trimming of non-crosslinked PG sidechains and catalyze the cleavage of the bond between meso-diaminopimelic acid (meso-Dap) and d-alanine, thus converting a PG tetrapeptide into a PG tripeptide. They are known to be cell-shape-determining enzymes, because deletion of the corresponding genes results in mutant strains that have lost the normal helical phenotype and instead possess a straight-rod morphology. In this work, we report two approaches directed towards the synthesis of the tripeptide substrate Ac-iso-d-Glu-meso-oxa-Dap-d-Ala, which serves as a mimic of the terminus of an non-crosslinked PG tetrapeptide substrate. The isosteric analogue meso-oxa-Dap was utilized in place of meso-Dap to simplify the synthetic procedure. The more efficient synthesis involved ring opening of a peptide-embedded aziridine by a serine-based nucleophile. A branched tetrapeptide was also prepared as a mimic of the terminus of a crosslinked PG tetrapeptide. We used MS analysis to demonstrate that the tripeptide serves as a substrate for both Csd6 and Pgp2 and that the branched tetrapeptide serves as a substrate for Pgp2, albeit at a significantly slower rate.

Preserving Insulin Secretion in Diabetes by Inhibiting VDAC1 Overexpression and Surface Translocation in ß Cells.

Type 2 diabetes (T2D) develops after years of prediabetes during which high glucose (glucotoxicity) impairs insulin secretion. We report that the ATP-conducting mitochondrial outer membrane voltage-dependent anion channel-1 (VDAC1) is upregulated in islets from T2D and non-diabetic organ donors under glucotoxic conditions. This is caused by a glucotoxicity-induced transcriptional program, triggered during years of prediabetes with suboptimal blood glucose control. Metformin counteracts VDAC1 induction. VDAC1 overexpression causes its mistargeting to the plasma membrane of the insulin-secreting ß cells with loss of the crucial metabolic coupling factor ATP. VDAC1 antibodies and inhibitors prevent ATP loss. Through direct inhibition of VDAC1 conductance, metformin, like specific VDAC1 inhibitors and antibodies, restores the impaired generation of ATP and glucose-stimulated insulin secretion in T2D islets. Treatment of db/db mice with VDAC1 inhibitor prevents hyperglycemia, and maintains normal glucose tolerance and physiological regulation of insulin secretion. Thus, ß cell function is preserved by targeting the novel diabetes executer protein VDAC1.

Effect of essential oils incorporated edible film on quality and storage stability of chicken patties at refrigeration temperature (4 ± 1 °C).

A blend of oregano and thyme essential oils (EOs) incorporated edible film was evaluated to improve the storage quality of chicken meat patties. Several preliminary trials were carried out to optimize the levels of bio-polymer to obtained desired edible film as a carrier and blend of EOs as bio preservatives. Preliminary studies indicated that 1.5% (w/v) solution of carrageenan as bio-polymer and 0.10% (v/v) oregano with 0.15% (v/v) thyme EOs in blend form as antimicrobial were suitable. Chicken meat patties wrapped with edible film incorporated with aforementioned EOs, packaged aerobically were stored at refrigeration temperature (4 ± 1 °C) for further studies. Results of refrigeration storage, showed that control samples had significantly higher pH and thiobarbituric acid reacting substances value than EOs treated products. There were significantly lower microbial counts observed in treatment samples (with EOs) and found well within permissible limit as compared to control. All the treatment samples showed lower or comparable flavour score in regard with control. It was found that shelf-life of chicken meat patties increased significantly (P < 0.05) during refrigerated storage and showed acceptable quality up to storage period of 30 days.

Activation of the Orphan G Protein-Coupled Receptor GPR27 by Surrogate Ligands Promotes ß-Arrestin 2 Recruitment.

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in ß-arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of ß-arrestin 2 to a GPR27V2 chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [N-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro-N-{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a N-phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology ß-arrestin 2 assay, imaging of green fluorescent protein-tagged ß-arrestin 2, and PathHunter ß-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.

Evaluation of anti-oxidant and anti-microbial activity of various essential oils in fresh chicken sausages.

The present study was undertaken to evaluate antimicrobial and antioxidant effect of essential oils on the quality of fresh (raw, ready to cook) chicken sausages. Several preliminary trials were carried out to optimize the level of four essential oils viz., clove oil, holybasil oil, thyme oil and cassia oil and these essential oils were incorporated at 0.25, 0.125, 0.25 and 0.125%, respectively in fresh chicken sausages. Quality evaluation and detailed storage stability studies were carried out for fresh chicken sausages for 20 days at refrigeration temperature (4 ± 1 °C). Refrigerated storage studies revealed that TBARS of control was significantly higher than treatment products whereas, total phenolics and DPPH activity was significantly lower in control. Among treatments, clove oil products had significantly lower TBARS but higher total phenolic content and DPPH activity followed by cassia oil, thyme oil and holybasil oil products. Microbial count of essential oil incorporated products were significantly lower than control and remained well below the permissible limit of fresh meat products (log107 cfu/g). Cassia oil products were observed with better anti-microbial characteristics than clove oil products at 0.25% level of incorporation, whereas, thyme oil products were better than holy basil oil products at 0.125% level. Storage studies revealed that clove oil (0.25%), holy basil oil (0.125%), cassia oil (0.25%) and thyme oil (0.125%) incorporated aerobically packaged and refrigerated fresh chicken sausages had approx. 4-5, 2-3, 5-6 and 2-3 days longer shelf life than control, respectively.

Anti-diabetic action of all-trans retinoic acid and the orphan G protein coupled receptor GPRC5C in pancreatic ß-cells.

Pancreatic islets express high levels of the orphan G-protein coupled receptor C5C (GPRC5C), the function of which remains to be established. Here we have examined the role of GPRC5C in the regulation of insulin secretion and ß-cell survival and proliferation using human and mouse pancreatic islets. The expression of GPRC5C was analysed by RNA-sequencing, qPCR, western blotting and confocal microscopy. Insulin secretion and cell viability were determined by RIA and MTS assays, respectively. GPRC5C mRNA expression and protein level were reduced in the islets from type-2 diabetic donors. RNA sequencing in human islets revealed GPRC5C expression correlated with the expression of genes controlling apoptosis, cell survival and proliferation. A reduction in Gprc5c mRNA and protein expression was observed in islets isolated from old mice (>46 weeks of age) compared to that in islets from newborn (<3 weeks) mice. Down-regulation of Gprc5c led to both moderately reduced glucose-stimulated insulin release and also reduced cAMP content in mouse islets. Potentiation of glucose-stimulated insulin secretion concomitant with enhanced islet cAMP level by all-trans retinoic acid (ATRA) was attenuated upon Gprc5c-KD. ATRA also increased [Ca+2]i in Huh7-cells. Gprc5c over expression in Huh7 cells was associated with increased ERK1/2 activity. Gprc5c-KD in clonal MIN6c4 cells reduced cell proliferation and in murine islets increased apoptosis and the sensitivity of primary islet cells to a cocktail of pro-apoptotic cytokines. Our results demonstrate that agents activating GPRC5C represent a novel modality for the treatment and/or prevention of diabetes by restoring and/or maintaining functional ß-cell mass.

Adhesion G Protein-Coupled Receptor G1 (ADGRG1/GPR56) and Pancreatic ß-Cell Function.

CONTEXT: Adhesion G protein-coupled receptor (GPCR)-G1 (ADGRG1) is the most abundant GPCR in human pancreatic islets, but its role in islet function is unclear. OBJECTIVE: Investigate how ADGRG1 expression and activation by its ligand, collagen III, impacts ß-cell function in normal and type 2 diabetic (T2D) islets. DESIGN: Genes associated with the ADGRG1 in human islets was probed by RNA-sequencing of human pancreatic islet isolated from cadaveric donors, followed by functional studies on ß-cell proliferation, apoptosis, and insulin secretion in human and mouse islets and in INS-1 cells. MAIN OUTCOME MEASURES: Changes in ß-cell gene expression, proliferation, apoptosis, and insulin secretion were quantified by RNA-sequencing, qPCR, Thymidine incorporation, Western blotting, and RIA, respectively. RESULTS: ADGRG1 is the most abundant GPCR mRNA in both human and mouse islets, and its expression in human islets strongly correlates with genes important for ß-cell function and T2D risk. The expression of ADGRG1 was reduced in islets of T2D donors, in db/db mouse islets, and in isolated human islets exposed to chronic hyperglycemia. Beneficial effects of collagen type III on ß-cell function via activation of the cAMP/protein kinase A pathway, suppression of RhoA and caspase-3 activity, increased ß-cell viability, and proliferation were abolished when ADGRG1 was down-regulated in ß-cells. CONCLUSIONS: We demonstrate a mechanistic link between ADGRG1 expression and ß-cell function. Pharmacological agents that promote expression or activation of the ADGRG1 receptor may represent a novel approach for the treatment of T2D.

Preparation and storage stability of meat spread developed from spent hens.

AIM: The present study was carried out to develop a meat spread as a healthier alternative to already existing meat products utilizing undervalued spent hen meat to add a new dimension to meat products. MATERIALS AND METHODS: Carcasses were processed within 30 min of slaughter and conditioned at 4±1°C for about 24 h and then braised along with other ingredients to get the final product. The products were evaluated for proximate composition, peroxide values, pH, microbiological, and sensory qualities as per standard procedures. RESULTS: The mean percent values for moisture, crude protein, ether extract, and total ash content of developed product were 58.75±0.32, 9.12±0.44, 11.19±0.16, and 2.35±0.17, respectively. No significant difference was observed for mean coliform and the yeast and mold counts with the progression of storage period, but samples differed significantly for mean pH, thiobarbituric acid and total viable plate count during storage of meat spread. A progressive decline in mean sensory scores was recorded along with the increase in storage time. CONCLUSION: The meat spread was found to be a good alternative to process the underutilized spent hens for its efficient utilization for product development.

GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion.

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Downregulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and b-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.

A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets.

Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.

Congenital ranula in a newborn: a rare presentation.

Ranulas are cystic lesions in the floor of the mouth. Case reports published worldwide have been very few. They are formed either as retention cyst or as pseudocyst due to extravasation of mucus in the surrounding tissue. We report the case of a full term female neonate with a congenital ranula in the floor of mouth on left side. The swelling caused no discomfort or complication and hence no immediate intervention was required. The ranula was treated by aspiration using a wide bore needle and did not recur on 4 months follow up. Other methods of treatment include excision of ranula, marsupialization, cryosurgery, sclerotherapy. As many congenital cysts resolve or rupture spontaneously, they should be observed for potential resolution for several months in uncomplicated cases.

WITHDRAWN: Potential link between alpha 1 anti-trypsin and PAR-2 in the prevention of beta cell dysfunction(☆).

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Ceftriaxone-vancomycin drug toxicity reduction by VRP 1020 in Mus musculus mice.

Drug toxicity is a common cause of liver injury and kidney failure. This study was designed to elucidate whether administration of high doses of Ceftriaxone or Vancomycin induce oxidative stress in liver as well as kidney, and to investigate the protective effects of VRP 1020 with fixed dose combination of ceftriaxone-vancomycin (Immunox-V). Twenty four Mus musculus mice (weighing 30 +/- 5 g) were divided into four groups containing six mice in each group. The activities of antioxidant enzymes such as superoxide dismutase, catalase and the level of malonaldialdehyde, as an marker of lipid per oxidation, were measured to evaluate oxidative stress in homogenates of the liver and renal tissue. Ceftriaxone or vancomycin administration significantly increased malonaldialdehyde levels (p < 0.001) but significant decreased in superoxide dismutase (p<0.01) and catalase (p<0.001) activities. Co-administration of VRP 1020 with FDC of Immunox-V injections caused significantly decreased malonaldialdehyde levels (p< 0.001) and increased superoxide dismutase (p<0.01) and catalase (p<0.001) activities in liver and renal tissue when compared with other treated groups. Similarly, the levels of extracellular antioxidant (Creatinine and Uric acid) were found to be significant lowered in Immunox-V treated group when compared to ceftriaxone or vancomycin alone treated group. These results indicate that chemical mediated technology of VRP 1020 with fixed dose combination of Immunox-V can prevent drug induced nephrotoxicity and oxidative stress which protects liver injury as well as renal tissue damage by reducing reactive oxygen species which improve the activities of free radical scavenging enzymes.

Protocol for improved extraction and PCR amplification of genomic DNA from liverwort, Plagiochasma appendiculatum.

A simplest method was followed for isolation of high quality genomic DNA form thallus of Plagiochasma appendiculatum that contained large quantities of polyphenols, terpenoids, tannins, contamination of high amount of RNA and polysaccharides. The method involved a modification of CTAB procedure using PVP (1%) and LiCl (4M) solution to remove polyphenols and RNA and some other binding proteins. The present protocol was found suitable for restriction enzyme digestion and random amplified polymorphic DNA (RAPD) analysis.

Fixed dose combination of cefepime plus amikacin prevent oxidative stress in liver of Mus musculus mice.

Oxidative stress and free radical are causative factors for aminoglycoside induced tissue injury. The objective of present study was to evaluate effect of fixed dose combination of cefepime + amikacin on antioxidant enzymes such as superoxide dismutase (SOD), catalase along with malonaldialdehyde (MDA) levels in liver of Mus musculus mice. Our findings showed that the activities of the antioxidant enzymes such as superoxide dismutase (p<0.001, 62.2%), catalase (p<0.05, 47.5%) were significantly lowered along with increase in the MDA levels (66.0%) after the single treatment of amikacin as compared to control group. A significant improvement in the activities of superoxide dismutase and catalase along with decrease in MDA levels were observed in fixed dose combination of cefepime plus amikacin treated groups as compared to amikacin alone treated group. These findings suggest that the fixed dose combination therapy of cefepime + amikacin (Potentox) show significant free radical scavenging property which may contribute to decrease in aminoglycoside induced liver injury.

Benefits of external beam irradiation for peripheral arterial bypass: preliminary report on a phase I study.

PURPOSE: To perform a Phase I study to determine the safety and feasibility of using external beam radiotherapy to prevent neointimal hyperplasia in patients after surgical bypass of occluded infrainguinal arteries. METHODS AND MATERIALS: All patients undergoing operative infrainguinal bypass for chronic ischemia were eligible for enrollment, although those requiring a prosthetic graft were preferentially considered. Immediately after bypass, the distal anastomosis was marked with clips, and the baseline anatomy of the anastomosis was documented with an intraoperative angiogram. The distal anastomotic site and 2 cm of surrounding tissues were irradiated to a total dose of 30 Gy, delivered in 10 fractions. The first dose was given within 48 h of surgery. RESULTS: Twenty-one patients were enrolled in this study. No anastomotic or wound problems or any other short-term complications of the treatment developed. However, at a mean follow-up of 10 months (range 3-18), 12 (57%) of the 21 grafts had occluded. Angiography was performed in 2 patients after successful thrombolysis and demonstrated normal anastomoses without residual stenosis. Evidence of stenosis at the irradiated anastomosis was seen in only 1 of the 21 patients by ongoing ultrasound surveillance. CONCLUSION: Fractionated external irradiation to a total dose of 30 Gy delivered to the distal surgical anastomosis immediately after operative bypass has no short-term complications and was associated with an apparently low rate of intimal hyperplasia. However, any possible gains made by reducing the neointimal hyperplasia at the site of anastomosis were significantly diminished by the high frequency of thrombotic events.