Comparative Evaluation of Salivary Parameters in Tobacco Substance Abusers.

BACKGROUND: Tobacco use by youth is ever-demanding, and it is increasingly distributed not only in India but also globally. Saliva is a complex oral bio-fluid, freely available, performing absolute tasks for maintaining oral health and homeostasis. It contains a plethora of significant constituents such as proline-rich proteins (PRPs), immunoglobulins, IgA, enzymes lysozyme, lactoferrin, peroxidases, amylase, etc. The basic ecological balance of the oral cavity is stabilized via salivary clearance by reduced aggregation and adherence of microorganisms by direct microbial activity. This balance of oral activity is also done by indirect mechanisms by immunological as well as non-immunological means and also by effectively regulating salivary pH flow rate. This institutional observational study was planned to assess and compare salivary parameters (pH, salivary flow rate), total proteins, α-amylase, calcium, phosphate, and IgA, of unstimulated whole saliva of both tobacco abusers and tobacco non-users. METHODS: The Study consisted of 270 participants (Tobacco habit) group, n = 135 and Control (Healthy) group, n = 135 and were in the age range of 20-50 years. They were assessed for oral health status, followed by the analysis of salivary pH, flow rate, total proteins, amylase, calcium, phosphates, and IgA of unstimulated whole saliva. RESULTS: Comparative evaluation of salivary parameters among groups found that varying tobacco abusers had increased salivary amylase, protein levels, and phosphate whereas decreased salivary pH, flow rate, IgA, and in the whole unstimulated saliva samples than those of non-tobacco users. This difference among groups was statistically significant. (p < 0.05), and calcium levels were not altered significantly. CONCLUSIONS: This study concludes that salivary parameters are altered in tobacco abusers when compared to those of non-abusers, and it was more significant in smokeless tobacco abusers than in any other form of tobacco abuse.

The cholinesterase and C-reactive protein score is a potential predictor of pseudoaneurysm formation after pancreaticoduodenectomy in patients with soft pancreas.

BACKGROUND: Pseudoaneurysm (PA) rupture after pancreaticoduodenectomy (PD) is a life-threatening complication. Most PA cases originate from postoperative pancreatic fistulas (POPFs). Although several risk factors for POPF have been identified, specific risk factors for PA formation remain unclear. Therefore, we retrospectively analyzed PD cases with soft pancreas and proposed a novel strategy for early detection of PA formation. METHODS: Overall, 120 patients underwent PD between 2010 and 2020 at our institution; of these, 65 patients with soft pancreas were enrolled. We evaluated the clinicopathological factors influencing PA formation and developed a risk score to predict PA formation. RESULTS: In total, 11 of the 65 patients developed PAs (PA formation group: PAG), and 8 of these 11 PAs ruptured. The median time to PA formation was 15 days, with a minimum of 5 days. The PAG was significantly older than the non-PA formation group, were predominantly men, and had comorbid diabetes mellitus. Pre- and intra-operative findings were similar between the two groups. Importantly, no significant differences were found in postoperative drain amylase levels and total drain amylase content. Cholinesterase and C-reactive protein (CRP) levels on postoperative day (POD) 3 were significantly different between the two groups. Multivariate analysis showed that cholinesterase ≤ 112 U/L and CRP ≥ 16.0 mg/dl on POD 3 were independent predictors of PA formation. CONCLUSIONS: Decreased cholinesterase and elevated CRP on POD 3 (Cho-C score) are useful predictors of PA formation in cases with soft pancreas. In such cases, periodic computed tomography evaluations and strict drain management are necessary to prevent life-threatening hemorrhage.

Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

BACKGROUND: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. RESULTS: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. CONCLUSIONS: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

Early postoperative risk stratification in patients with pancreatic fistula after pancreaticoduodenectomy.

BACKGROUND: Early stratification of postoperative pancreatic fistula according to severity and/or need for invasive intervention may improve outcomes after pancreaticoduodenectomy. This study aimed to identify the early postoperative variables that may predict postoperative pancreatic fistula severity. METHODS: All patients diagnosed with biochemical leak and clinically relevant-postoperative pancreatic fistula based on drain fluid amylase >300 U/L on the fifth postoperative day after pancreaticoduodenectomy were identified from a consecutive cohort from Birmingham, UK. Demographics, intraoperative parameters, and postoperative laboratory results on postoperative days 1 through 7 were retrospectively extracted. Independent predictors of clinically relevant-postoperative pancreatic fistula were identified using multivariable binary logistic regression and converted into a risk score, which was applied to an external cohort from Verona, Italy. RESULTS: The Birmingham cohort had 187 patients diagnosed with postoperative pancreatic fistula (biochemical leak: 99, clinically relevant: 88). In clinically relevant-postoperative pancreatic fistula patients, the leak became clinically relevant at a median of 9 days (interquartile range: 6-13) after pancreaticoduodenectomy. Male sex (P = .002), drain fluid amylase-postoperative day 3 (P < .001), c-reactive protein postoperative day 3 (P < .001), and albumin-postoperative day 3 (P = .028) were found to be significant predictors of clinically relevant-postoperative pancreatic fistula on multivariable analysis. The multivariable model was converted into a risk score with an area under the receiver operating characteristic curve of 0.78 (standard error: 0.038). This score significantly predicted the need for invasive intervention (postoperative pancreatic fistula grades B3 and C) in the Verona cohort (n = 121; area under the receiver operating characteristic curve: 0.68; standard error = 0.06; P = .006) but did not predict clinically relevant-postoperative pancreatic fistula when grades B1 and B2 were included (area under the receiver operating characteristic curve 0.52; standard error = 0.07; P = .802). CONCLUSION: We developed a novel risk score based on early postoperative laboratory values that can accurately predict higher grades of clinically relevant-postoperative pancreatic fistula requiring invasive intervention. Early identification of severe postoperative pancreatic fistula may allow earlier intervention.

The effects of eugenol on histological, enzymatic, and oxidative parameters in the major salivary glands and pancreas of healthy male Wistar rats.

OBJECTIVE: We evaluated the effects of eugenol on histological, enzymatic, and oxidative parameters in the pancreas, parotid, submandibular, and sublingual glands of healthy male rats. DESIGN: Twenty-four adult Wistar rats were assigned into four groups (n = 6/group). Control rats received 2% Tween-20 (eugenol vehicle), whereas the other animals received 10, 20, and 40 mg kg-1 eugenol through gavage daily for 60 d. Major salivary and pancreatic glands were weighed and preserved fixed for microscopic analysis and frozen for in vitro assays. RESULTS: Eugenol did not alter glands' weight and serum amylase activity regardless of the concentration. The highest dose of eugenol caused an increase in pancreatic amylase activity and a reduction of lipase activity from serum and pancreas. Eugenol at 40 mg kg-1 diminished the activity of SOD and FRAP in the submandibular gland and CAT and FRAP in the sublingual gland. However, it did not exert any effect on GST regardless of the gland. Additionally, 40 mg kg-1 eugenol increased MDA levels in pancreatic, parotid, and submandibular glands and NO levels in the sublingual. The concentrations of eugenol induced distinct responses in the glands regarding the activity of Na+/K+, Mg2+, and total ATPase activity. They also affected histomorphometrical and histochemistrical parameters in the submandibular gland only. CONCLUSIONS: Results indicated that 40 mg kg-1 eugenol altered most of the biochemical and oxidatived parameters of digestive glands. Only submandibular glands presented histological changes after eugenol exposure suggesting potential implications for its function.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

The Distinctive Permutated Domain Structure of Periplasmic α-Amylase (MalS) from Glycoside Hydrolase Family 13 Subfamily 19.

Periplasmic α-amylase MalS (EC. 3.2.1.1), which belongs to glycoside hydrolase (GH) family 13 subfamily 19, is an integral component of the maltose utilization pathway in Escherichia coli K12 and used among Ecnterobacteriaceae for the effective utilization of maltodextrin. We present the crystal structure of MalS from E. coli and reveal that it has unique structural features of circularly permutated domains and a possible CBM69. The conventional C-domain of amylase consists of amino acids 120-180 (N-terminal) and 646-676 (C-terminal) in MalS, and the whole domain architecture shows the complete circular permutation of C-A-B-A-C in domain order. Regarding substrate interaction, the enzyme has a 6-glucosyl unit pocket binding it to the non-reducing end of the cleavage site. Our study found that residues D385 and F367 play important roles in the preference of MalS for maltohexaose as an initial product. At the active site of MalS, ß-CD binds more weakly than the linear substrate, possibly due to the positioning of A402. MalS has two Ca2+ binding sites that contribute significantly to the thermostability of the enzyme. Intriguingly, the study found that MalS exhibits a high binding affinity for polysaccharides such as glycogen and amylopectin. The N domain, of which the electron density map was not observed, was predicted to be CBM69 by AlphaFold2 and might have a binding site for the polysaccharides. Structural analysis of MalS provides new insight into the structure-evolution relationship in GH13 subfamily 19 enzymes and a molecular basis for understanding the details of catalytic function and substrate binding of MalS.

The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Microscopic assessment of the degradation of millet starch granules by endogenous and exogenous enzymes during mashing.

The high gelatinization temperature (GT) of millet starch prevents the usage of infusion or step mashes as an effective means to generate fermentable sugars (FS) in brewing because the malt amylases lack thermostability at GT. Here, we investigate processing modifications to determine if millet starch can be efficiently degraded below GT. We determined that producing finer grists through milling did not introduce enough granule damage to markedly change gelatinization characteristics, though there was improved liberation of the endogenous enzymes. Alternatively, exogenous enzyme preparations were added to investigate their ability to degrade intact granules. At the recommended dosages (0.625 µL/g malt), significant FS concentrations were observed, although at lower concentrations and with a much-altered profile than possible with a typical wort. When exogenous enzymes were introduced at high (10×) addition rates, significant losses of granule birefringence and granule hollowing were observed well below GT, suggesting these exogenous enzymes can be utilized to digest millet malt starch below GT. The exogenous maltogenic α-amylase appears to drive the loss of birefringence, but more research is needed to understand the observed predominate glucose production.

Anti-Cholinesterase and Anti-α-Amylase Activities and Neuroprotective Effects of Carvacrol and p-Cymene and Their Effects on Hydrogen Peroxide Induced Stress in SH-SY5Y Cells.

Several researchers have demonstrated the health and pharmacological properties of carvacrol and p-cymene, monoterpenes of aromatic plants. This study investigated these compounds' possible anti-cholinesterase, anti-α-amylase, and neuroprotective effects. We evaluated the anti-acetylcholinesterase and anti-α-amylase activities at different concentrations of the compounds. The maximum non-toxic dose of carvacrol and p-cymene against SH-SY5Y neuroblastoma cells was determined using an MTT assay. The neuroprotective effects of the compounds were evaluated on H2O2-induced stress in SH-SY5Y cells, studying the expression of caspase-3 using Western blotting assays. Carvacrol showed inhibitory activities against acetylcholinesterase (IC50 = 3.8 µg/mL) and butyrylcholinesterase (IC50 = 32.7 µg/mL). Instead, the anti-α-amylase activity of carvacrol resulted in an IC50 value of 171.2 µg/mL After a pre-treatment with the maximum non-toxic dose of carvacrol and p-cymene, the expression of caspase-3 was reduced compared to cells treated with H2O2 alone. Carvacrol and p-cymene showed in vitro anti-enzymatic properties, and may act as neuroprotective agents against oxidative stress. Further studies are necessary to elucidate their possible use as coadjutants in preventing and treating AD in diabetic patients.

Exploring the interactive mechanism of acarbose with the amylase SusG in the starch utilization system of the human gut symbiont Bacteroides thetaiotaomicron through molecular modeling.

The α-amylase, SusG, is a principal component of the Bacteroides thetaiotaomicron (Bt) starch utilization system (Sus) used to metabolize complex starch molecules in the human gastrointestinal (GI) tract. We previously reported the non-microbicidal growth inhibition of Bt by the acarbose-mediated arrest of the Sus as a potential therapeutic strategy. Herein, we report a computational approach using density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation to explore the interactive mechanism between acarbose and SusG at the atomic level in an effort to understand how acarbose shuts down the Bt Sus. The docking analysis reveals that acarbose binds orthosterically to SusG with a binding affinity of -8.3 kcal/mol. The MD simulation provides evidence of conformational variability of acarbose at the active site of SusG and also suggests that acarbose interacts with the main catalytic residues via a general acid-base double-displacement catalytic mechanism. These results suggest that small molecule competitive inhibition against the SusG protein could impact the entire Bt Sus and eliminate or reduce the system's ability to metabolize starch. This computational strategy could serve as a potential avenue for structure-based drug design to discover other small molecules capable of inhibiting the Sus of Bt with high potency, thus providing a holistic approach for selective modulation of the GI microbiota.

Evaluation of Biological Activities of Twenty Flavones and In Silico Docking Study.

This work aimed to evaluate the biological activities of 20 flavones (M1 to M20) and discuss their structure-activity relationships. In vitro assays were established to assess their numerous biological activities (anti-α-amylase, anti-acetylcholinesterase, anti-xanthine oxidase, anti-superoxide dismutase, and anticancer cell lines (HCT-116, MCF7, OVCAR-3, IGROV-1, and SKOV-3 cells lines)). An in silico docking study was also established in order to find the relationship between the chemical structure and the biological activities. In vitro tests revealed that M5 and M13 were the most active in terms of anti-α-amylase activity (IC50 = 1.2 and 1.4 µM, respectively). M17 was an inhibitor of xanthine oxidase (XOD) and performed better than the reference (allopurinol), at IC50 = 0.9 µM. M7 presented interesting anti-inflammatory (IC50 = 38.5 µM), anti-supriode dismutase (anti-SOD) (IC50 = 31.5 µM), and anti-acetylcholinesterase (IC50 = 10.2 µM) activities. Those abilities were in concordance with its high scavenging activity in antioxidant ABTS and DPPH assays, at IC50 = 6.3 and 5.2 µM, respectively. Selectivity was detected regarding cytotoxic activity for those flavones. M1 (IC50 = 35.9 µM) was a specific inhibitor to the MCF7 cancer cell lines. M3 (IC50 = 44.7 µM) and M15 (IC50 = 45.6 µM) were particularly potent for the OVCAR-3 cell line. M14 (IC50 = 4.6 µM) contributed more clearly to inhibiting the colon cancer cell line (HCT116). M7 (IC50 = 15.6 µM) was especially active against the ovarian SKOV human cancer cell line. The results of the biological activities were supported by means of in silico molecular docking calculations. This investigation analyzed the contribution of the structure-activity of natural flavones in terms of their biological properties, which is important for their future application against diseases.

Feeding amylolytic and proteolytic exogenous enzymes: Effects on nutrient digestibility, ruminal fermentation, and performance in dairy cows.

Exogenous enzymes are added to diets to improve nutrient utilization and feed efficiency. A study was conducted to evaluate the effects of dietary exogenous enzyme products with amylolytic (Amaize, Alltech) and proteolytic (Vegpro, Alltech) activity on performance, excretion of purine derivatives, and ruminal fermentation of dairy cows. A total of 24 Holstein cows, 4 of which were ruminally cannulated (161 ± 88 d in milk, 681 ± 96 body weight, and 35.2 ± 5.2 kg/d of milk yield), were blocked by milk yield, days in milk, and body weight, and then distributed in a replicated 4 × 4 Latin square design. Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation and the last 7 d were used for data collection. Treatments were as follows: (1) control (CON) with no feed additives, (2) amylolytic enzyme product added at 0.5 g/kg diet dry matter (DM; AML), (3) amylolytic enzyme product at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.2 g/kg of diet DM (low level; APL), and (4) amylolytic enzyme products added at 0.5 g/kg diet DM and proteolytic enzyme product at 0.4 g/kg of diet DM (high level; APH). Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). Differences between treatments were analyzed by orthogonal contrasts: CON versus all enzyme groups (ENZ); AML versus APL+APH; and APL versus APH. Dry matter intake was not affected by treatments. Sorting index for feed particles with size <4 mm was lower for ENZ group than for CON. Total-tract apparent digestibility of DM and nutrients (organic matter, starch, neutral detergent fiber, crude protein, and ether extract) were similar between CON and ENZ. Starch digestibility was greater in cows fed APL and APH treatments (86.3%) compared with those in the AML group (83.6%). Neutral detergent fiber digestibility was greater in APH cows compared with those in the APL group (58.1 and 55.2%, respectively). Ruminal pH and NH3-N concentration were not affected by treatments. Molar percentage of propionate tended to be greater in cows fed ENZ treatments than in those fed CON. Molar percentage of propionate was greater in cows fed AML than those fed the blends of amylase and protease (19.2 and 18.5%, respectively). Purine derivative excretions in urine and milk were similar in cows fed ENZ and CON. Uric acid excretion tended to be greater in cows consuming APL and APH than in those in the AML group. Serum urea N concentration tended to be greater in cows fed ENZ than in those fed CON. Milk yield was greater in cows fed ENZ treatments compared with CON (32.0, 33.1, 33.1, and 33.3 kg/d for CON, AML, APL, and APH, respectively). Fat-corrected milk and lactose yields were higher when feeding ENZ. Feed efficiency tended to be greater in cows fed ENZ than in those fed CON. Feeding ENZ benefited cows' performance, whereas the effects on nutrient digestibility were more pronounced when the combination of amylase and protease was fed at the highest dose.

Optimal timing of free total rhubarb anthraquinones on immune regulation in rats with severe acute pancreatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhubarb is the peeled and dried root of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill. Free total rhubarb anthraquinones (FTRAs) isolated and extracted from rhubarb display the beneficial effects of anti-inflammation and immunological modulation. The timing of immune regulation is a major problem in the immunotherapy for severe acute pancreatitis (SAP). several studies reported that FTRAs could reduce systemic inflammatory responses by inhibiting early immune overactivity in the gut in rats with SAP. But, the optimal timing of rhubarb and FTRAs administration is not clear in clinical practice. Therefore, the time window for the best efficacy of rhubarb and FTRAs in the treatment of SAP patients should be further elucidated. AIM OF THE STUDY: The main purpose of the present study was to evaluate the efficacy and optimal timing of immune modulation with FTRAs in the treatment of SAP in rats. MATERIALS AND METHODS: FTRAs (22.5, 45 and 90 mg/kg), Rhubarb (RHU) (900 mg/kg, positive control) or normal saline (vehicle control) were initiated at 0 (immediately), 48 and 72 h every 12 h for three times in total. The therapeutic effects of FTRAs and RHU on pancreas and intestinal tissues injury, secondary infection with pseudomonas aeruginosa (PA), amylase, lipase, D-lactic acid (DLA), endotoxin (ET), proinflammatory and anti-inflammatory cytokines, macrophages, dendritic cells and regulatory T cells (Tregs) in the blood, small intestine and/or mesenteric lymph node (MLN) were determined in rats with SAP after treatment. RESULTS: The results showed that administration of FTRAs at 0 h was superior to 48 h and 72 h, which significantly protected the injury of pancreas and intestinal tissues, reduced the mortality induced by secondary infection with PA, decreased the levels of amylase, lipase, DLA, ET, tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), IL-6, IL-8, IL-18 and Tregs, and increased the levels of IL-4, sTNF-αR, macrophages and dendritic cells, secretary immunoglobulin A (SIgA) in the blood and/or small intestinal tissues in rats with SAP. CONCLUSIONS: In conclusion, our studies indicate that the treatment window of FTRAs for SAP is within 48 h of development, administration of FTRAs at the early stage (0 h, immune overreaction period) was the optimal time and superior to that of 48 h and 72 h for its therapeutic efficacy. The earlier the administration of FTRAs, the better the therapeutic efficacy. Therefore, our data may provide a scientific rationale for the clinical application and optimal timing of FTRAs in the treatment of SAP.

Predicting stress response and improved protein overproduction in Bacillus subtilis.

Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis, iJT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed iJT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like iJT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

Comparative Metabolomic Analysis of Moromi Fermented Using Different Aspergillus oryzae Strains.

Aspergillus oryzae (A. oryzae) is an important starter in the fermentation of koji and moromi. However, the effect of different A. oryzae strains on the quality of moromi has rarely been studied. For this reason, this study analyzed the physicochemical properties, enzyme activity, sensory quality, and metabolite profiles of moromi samples fermented using two strains (A. oryzae KCCM12012P (moromi-1) and KCCM12804P (moromi-2)), which were newly isolated from fermented soy foods, and compared them to those of a commercialized A. oryzae strain (control). Amino-type nitrogen contents of moromi-1 and moromi-2 samples were higher than that of control moromi, and their amylase and protease activities were also higher. Moreover, metabolite profiles of moromi were significantly altered according to strains. In particular, the levels of many amino acids, peptides, nucleotides, and acidic compounds were altered, which resulted in changes in the sensory quality of moromi. Although volatile compounds were not investigated, the results suggested that the quality of moromi was significantly different for newly isolated strains, especially A. oryzae KCCM12804P, and they were superior to the commercial strain in terms of taste-related substances. Therefore, these strains could be used as good starters to produce moromi and soy sauce with good sensory quality.

Bifidobacterium spp. and their metabolite lactate protect against acute pancreatitis via inhibition of pancreatic and systemic inflammatory responses.

Severe acute pancreatitis (SAP) is a critical illness characterized by a severe systemic inflammatory response resulting in persistent multiple organ failure and sepsis. The intestinal microbiome is increasingly appreciated to play a crucial role in modulation of AP disease outcome, but limited information is available about the identity and mechanism of action for specific commensal bacteria involved in AP-associated inflammation. Here we show that Bifidobacteria, particularly B. animalis, can protect against AP by regulating pancreatic and systemic inflammation in germ-free (GF) and oral antibiotic-treated (Abx) mouse models. Colonization by B. animalis and administration of its metabolite lactate protected Abx and GF mice from AP by reducing serum amylase concentration, ameliorating pancreatic lesions and improving survival rate after retrograde injection of sodium taurocholate. B. animalis relieved macrophage-associated local and systemic inflammation of AP in a TLR4/MyD88- and NLRP3/Caspase1-dependent manner through its metabolite lactate. Supporting our findings from the mouse study, clinical AP patients exhibited a decreased fecal abundance of Bifidobacteria that was inversely correlated with the severity of systemic inflammatory responses. These results may shed light on the heterogeneity of clinical outcomes and drive the development of more efficacious therapeutic interventions for AP, and potentially for other inflammatory disorders.

Characteristic Analysis of Soil-Isolated Bacillus velezensis HY-3479 and Its Antifungal Activity Against Phytopathogens.

During the investigation of beneficial agricultural microorganisms, a novel Bacillus strain was isolated. To isolate an effective microorganism that has antifungal activity, soil samples were collected from an agricultural field in the southern area of Pohang, Korea. One strain that had specificity on plant pathogens was analyzed. According to 16S rRNA sequencing, the isolated bacterium was identified as Bacillus velezensis and was designated as HY-3479. Few assays were taken to analyze the characteristics of the HY-3479 strain. In agar plate assay, HY-3479 showed antifungal effects on Colletotrichum acutatum, Cylindrocarpon destructans, Rhizoctonia solani, and Sclerotinia sclerotiorum. The strain also had various enzymatic activities including protease, amylase, and ß-1,3-glucanase, which were relatively higher than control strains. Metabolites study of strain HY-3479 was conducted by GC-MS analysis and the bacterium contained many plant growth promoters like 3-methyl-1-butanol, (R, R)-2,3-butanediol, acetoin, and benzoic acid which were not found in untreated TSB medium. In gene expression analysis, antifungal lipopeptide genes like srfc (surfactin) and ituD (iturin A) were highly produced in the HY-3479 strain compared to the control strain KCTC 13417. B. velezensis strain HY-3479 may be the candidate to be an effective microorganism in agriculture and become a beneficial biocontrol agent with plant growth-promoting activities.