The Effect of Neoglandin on the Activity of N-Acetyl-ß-D-Hexosaminidase in the Serum and Urine of Alcohol-Dependent Men.

Dietary supplementation of gamma-linolenic acid (GLA) in the form of a commercial drug neoglandin (containing GLA and vitamin E), in people following alcohol abuse allows bypassing of the ineffective delta-6-desaturase system involved in the transformation of linoleic acid into GLA. Determination of the activity of N-acetyl-ß-D-hexosaminidase (HEX) in the serum and urine reflects neoglandin action on the catabolism of glycoconjugates and the functioning of liver and kidneys in people following alcohol abuse. MATERIAL AND METHODS: The serum and urine were collected from men with alcohol dependence, treated (n = 31, age 33.16 ± 9.72 years) and not treated (n = 50, age 35.46 ± 11.37 years) with neoglandin. HEX activity were assayed in the supernatants by the colorimetric method, with the p-nitrophenyl derivative of sugar as substrate. RESULTS: Our study on alcoholic men not treated with neoglandin indicates a significantly higher concentration of the serum and urinary HEX activity (nKat/L) on day 1 compared to days 7, 10, 14 and 30 (p < 0.001). For days 14 and 30 (p < 0.01), the urinary HEX activity was expressed in µKat/kgCr. No significant differences were observed in the activity of serum (nKat/L) and urinary (nKat/L and µKat/kgCr) HEX in alcoholics during treatment with neoglandin compared to day 1 of neoglandin treatment. We found significantly different (p < 0.05) concentration of HEX activity (nKat/L) in serum of alcohol-dependent men treated with neoglandin compared to those not taking neoglandin on days 7, 10, 14 and 30 of treatment. The urinary concentration of HEX activity (nKat/L) on days 1, 4, 10 and 30 and HEX activity in µKat/kgCr on days 1, 4 and 7 it was significantly higher (p < 0.05) during the treatment of alcohol-dependence without the use of neoglandin as compared to alcoholics treated with neoglandin. We found a positive correlation between the amount of alcohol consumed and the urinary activity of HEX in the early phase after alcohol withdrawal and a lack of correlation between the HEX activity in serum and urine of alcohol-dependent men not treated with neoglandin. CONCLUSIONS: Neoglandin supplementation in alcoholic men significantly slows down the catabolism of glycoconjugates, thus reducing the effects of ethanol poisoning that are harmful to the kidneys. Neoglandin reduces the harmful effects of ethanol poisoning more on the kidneys than on the liver. The activity of HEX in the serum may be used in monitoring the treatment of alcoholism and whether alcohol reuse occurred during the therapy. In the early stages of alcohol withdrawal, urinary HEX activity can be used as a marker of the amount of alcohol consumed during previous alcohol abuse.

Comparison of the Effects of Laparoscopic Surgery and Traditional Open Surgery on Stone Clearance, Laboratory Indexes and Life Quality in Patients with Renal Calculi.

Objective: A case-control study was conducted to determine the effectiveness of laparoscopic surgery and traditional open surgery on stone clearance, laboratory indexes, and life quality in patients with renal calculi. Methods: During March 2017 to March 2022, 272 patients with complex renal calculi (CRC) cured in our hospital were assigned into control group (n = 136) and research group (n = 136) arbitrarily. The former accepted traditional open surgery, while the latter accepted laparoscopic surgery. The operation time, intraoperative blood loss, hospital stay, and time of getting out of bed were compared. The degree of postoperative incision pain was assessed by visual analogue scale (VAS). The life quality was assessed by the Comprehensive Assessment Questionnaire-74 (GQOL-74). The indexes of renal function and urine metabolism were measured. Then, the postoperative stone clearance rate and complications were calculated. Results: Operation time, blood loss intraoperatively, time out of bed, and hospitalization were all remarkably reduced in the research group, and the difference was statistically significant (P < 0.05). The complete stone clearance rates in study and control cohorts were 75.73% and 63.24%, respectively. The VAS scores were lessened after the operation. Compared with the two groups, the VAS scores of the research group were remarkably lower at 1 to 2 weeks and 1 and 3 months after the operation, and the difference was statistically significant (P < 0.05). One week after operation, the levels of ß 2-microglobulin (ß 2-MG), N-acetyl-ß-glucosaminidase (NAG), and renal injury molecule-1 (kidney injury molecule-1, Kim-1) in the research group were remarkably lower. The levels of urinary ß 2-MG, NAG, and KIM-1 in the research group were remarkably lower, and the difference was statistically significant (P < 0.05). One week after operation, the levels of urinary oxalic acid, uric acid, and urinary calcium lessened averagely. The levels of urinary oxalic acid, uric acid, and urinary calcium in the research group were lower, and the difference was statistically significant (P < 0.05). The quality-of-life scores were compared. One week after the operation, the scores of physical function, psychological function, social function, and material function were all augmented, and the difference was statistically significant (P < 0.05). The incidence of complications was 9.56% and 2.21%, respectively. The incidence of complications in the research group was lower, and the difference was statistically significant (P < 0.05). Conclusion: Laparoscopic surgery is successful when treating CRC, which is superior to invasive surgery in postoperative complications, stone clearance rate, improvement of postoperative renal function, and life quality. It is one of the ideal treatment methods for CRC. However, the role of open surgery when treating CRC cannot be ignored. This needs to be further confirmed by large samples of randomized controlled trials.

[Multicenter randomized study of bovhyalu-ronidase azoximer (Longidaza) in men after transuretral resection of the prostate].

INTRODUCTION: Transurethral resection of the prostate (TURP) is the gold standard of BPH surgical treatment. It is of current interest to search for medications that can reduce the incidence of complications after TURP. AIM: To evaluate the efficiency of Longidaza (rectal suppositories of 3000 IU) as part of combined therapy in order to prevent complications after TURP. MATERIALS AND METHODS: The study included 202 patients who underwent TURP in 3 hospitals. The patients were divided into 2 groups: main group - 96 men taking standard postoperative therapy with Longidaza rectal suppositories N 20; control group - 106 men - taking standard postoperative therapy (tamsulosin 30 days; fluoroquinolone 5 days). Follow-up included IPSS, urinalysis, urine culture, ultrasound examination of the prostate volume (PV), post void residual urine, uroflowmetry at 1,2,3,6 months after surgery. Average preoperative indices: IPSS 27 [23; 30], Qol 5 [4; 6], prostate volume (PV) 71+/-19cc (30-272 c), Qmax 7.5+/-2.5ml/s (1,3-18,7 ml/s). RESULTS: There was a significant improvement in IPSS, QoL, Qmax, PV, post void residual urine (PVR) compared to preoperative values during the entire observation period. There was no statistical difference between the main and control groups for these indexes in 6 months. In the main group had statistically lower incidence of bacteriuria at 3 (11% vs 17%) and 6 months (7% vs 17%), and leukocyturia at 3 (31% vs 46%) and 6 months of follow-up (20% vs 44%). Overall incidence of infectious complications and additional antibacterial drugs prescription was lower in the Longidaza group compared to the control group (17,7% vs 20,7%). Urethral strictures developed in 7 men in the main group, and 8 in the control group. CONCLUSION: Our results show that prescription of Longidaza significantly reduces the incidence of leukocyturia and bacteriuria postoperatively, decreasing the rate of infectious complications in men after TURP.

Inhibitory effects of cyanidin-3-O-glucoside in black soybean hull extract on RBL-2H3 cells degranulation and passive cutaneous anaphylaxis reaction in mice.

Black soybean hull extract (BSHE) exhibits a variety of biological activities. However, little is known about the effects of BSHE on immunoglobulin E (IgE)-mediated type I allergic reactions. The anti-allergic effect of BSHE was assessed with the degranulation assay using rat basophilic leukemia RBL-2H3 cells and the passive cutaneous anaphylaxis (PCA) reaction in mice. An active compound in BSHE was identified by ultra-performance liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry analysis. BSHE inhibited the release of ß-hexosaminidase and histamine in RBL-2H3 cells, and cyanidin-3-O-glucoside (C3G) was identified as one of its active compounds. Oral administering of 200 µmol/kg of C3G to IgE-sensitized mice prior to antigen injection suppressed the PCA reaction, as compared with control (p < 0.01). Intravenous administration of BSHE (C3G content, 5.4%) more strongly inhibited PCA responses at lower doses (100 mg/kg, p < 0.01) than oral administration (1,000 mg/kg, p = 0.059). Intravenous C3G also suppressed PCA response at a low dose (40 mg/kg, p < 0.05), showing the same trend as BSHE. This information can be useful to design appropriate formulations of anthocyanin-based drug products to suppress allergic reactions. This study provides evidence for the potential use of BSHE and C3G for the prevention or the treatment of type I allergies.

Influence of dietary Chlorella vulgaris and carbohydrate-active enzymes on growth performance, meat quality and lipid composition of broiler chickens.

Herein, we investigated the effect of Chlorella vulgaris as ingredient (10% of incorporation) in broiler diets, supplemented or not with 2 formulations of Carbohydrate-Active enZymes (CAZymes; Rovabio Excel AP and a mixture of recombinant CAZymes, composed by an exo-ß-glucosaminidase, an alginate lyase, a peptidoglycan N-acetylmuramic acid deacetylase and a lysozyme), on growth performance, meat quality, fatty acid composition, oxidative stability, and sensory traits. One hundred twenty 1-day-old Ross 308 male birds were randomly assigned to one of the 4 experimental diets (n = 30): corn-soybean meal-basal diet (control), basal diet with 10% C. vulgaris (CV), CV supplemented with 0.005% of a commercial CAZyme cocktail (Rovabio Excel AP), (CV + R), and CV supplemented with 0.01% of a 4-CAZyme mixture previously selected (CV + M) during the experimental period lasted from day 21 to day 35. Body weight gain and feed conversion rate of broilers were not affected by C. vulgaris but digesta viscosity increased more than 2-fold (P < 0.001) relative to the control. In addition, neither cooking loss, shear force, juiciness, flavor nor off-flavor was impaired by dietary treatments (P > 0.05). By contrast, the dietary C. vulgaris increased tenderness, yellowness (b∗) and total carotenoids in breast and thigh meats. However, no additional protective effect against lipid oxidation was observed in meat with the inclusion of microalga. Chlorella vulgaris, independently of CAZymes, had a minor impact on meat fatty acid composition but improved the proportion of some beneficial fatty acids. In summary, our data indicate a slight improvement of broiler meat quality and lipid nutritional value, without impairment of broilers' growth performance, thus supporting the usefulness of this microalga in poultry diets, up to this high level of incorporation. By contrast, the selected CAZyme mixtures used do not significantly improve the release of microalga nutrients in poultry diets, through the disruption of microalga cell wall, which warrants further research.

Inhibitory Effects of Green Asparagus Extract, Especially Phospholipids, on Allergic Responses in Vitro and in Vivo.

Asparagus (Asparagus officinalis L.) is one of the widely consumed vegetables. To investigate the mechanism underlying the anti-allergic responses of asparagus, we extracted different fractions from asparagus and measured their inhibitory effects on ß-hexosaminidase release in RBL-2H3 cells in vitro and an atopic dermatitis NC/Nga mouse model in vivo. The lipid fractions from asparagus were extracted with 50% ethanol, separated using chloroform by liquid-liquid phase separation, and fractionated by solid-phase extraction. Among them, acetone fraction (rich in glycolipid) and MeOH fraction (rich in phospholipid) markedly inhibited ß-hexosaminidase release from RBL-2H3 cells. In NC/Nga mice treated with picryl chloride, atopic dermatitis was alleviated following exposure to the 50% EtOH extract, acetone fraction, and methanol fraction. The inhibitory effects of asparagus fractions in vivo were supported by the significant decrease in serum immunoglobulin E (IgE) levels. The phospholipid fractions showed significantly better inhibitory effects, and phosphatidic acid from this fraction showed the best inhibitory effect on ß-hexosaminidase release. In mice challenged with ovalbumin (OVA), oral administration of asparagus extract and its fractions decreased the OVA-specific IgE level and total IgE, indicating that these effects may be partly mediated through the downregulation of antigen-specific IgE production. Taken together, the present study shows for the first time that asparagus extract and its lipid fractions could potentially mitigate allergic reactions by decreasing degranulation in granulocytes. Our study provides useful information to develop nutraceuticals and functional foods fortified with asparagus.

Use of glycerol and propylene glycol as additives in heat-treated goat colostrum.

This experiment aimed to evaluate the suitability of glycerol and propylene glycol to reduce microbial count and preserve immune properties in heat-treated goat colostrum. Colostrum samples from 11 goats were each divided into 9 aliquots. Different concentrations (2, 6, 10, and 14%; vol/vol) of either glycerol or propylene glycol were added to the aliquots. Phosphate buffer solution was added to one aliquot, which was set as the control (CG). After the respective additions, all colostrum samples were heat treated at 56°C for 1 h. After cooling, aerobic mesophilic bacteria were cultured. The samples were frozen until free fatty acid, IgG, IgA, and IgM concentrations and chitotriosidase activity were measured. No differences were found in aerobic mesophilic bacteria counts between either 10 or 14% glycerol and propylene glycol additives. These additions reduced bacterial count to a greater extent than CG, and 2 or 6% additions. Colostrum IgG concentration was not affected by either of the additives or their concentrations. The propylene glycol additive reduced IgA and IgM concentrations and chitotriosidase activity, compared with CG. Conversely, glycerol did not affect any of the studied immune variables. In conclusion, glycerol addition to goat colostrum before heat treatment is suitable to enhance bacterial reduction, whereas colostrum immune properties were not affected.

Sheep and goats raised in mixed flocks have diverse immune status around parturition.

Several physiological and metabolic changes take place in dairy ruminants around parturition (late pregnancy, parturition, and early lactation). Dairy species are genetically selected for their higher milk production compared with non-dairy species. This fact causes a constant stress that impairs the immune status of the animal, with consequences for its welfare and performance. In the present study, we assessed the immune status of high-yield dairy sheep and goats by quantifying IgG and IgM concentrations, as well as chitotriosidase (ChT) and complement system [total complement system (TC) and alternative complement pathway (AC)] activity in blood plasma around parturition. We also measured IgG and IgM concentrations and ChT activity in colostrum and milk during the first 40 d postpartum. The lowest blood IgG concentration was at parturition in both species. We detected no differences in blood IgG concentrations between species. Blood IgM concentrations were constant in both species throughout the study period. However, blood IgM concentrations were greater in sheep than in goats. Blood ChT activity was greater in goats than in sheep, and both species showed constant activity of this enzyme throughout the study period. We observed no differences in complement system (TC and AC) activity between sheep and goats. In addition, both TC and AC activity were constant in both species throughout the experiment. In general, IgG and IgM concentrations were greater in sheep colostrum than in goat colostrum, but these differences disappeared after d 4 (IgG) and d 3 (IgM) postpartum. In both species, the highest IgG and IgM concentrations were measured in colostrum, gradually decreasing during the first days postpartum. Chitotriosidase activity decreased in both species from colostrum to milk, although goats always showed greater ChT activity than sheep. Both sheep and goats seemed to be more susceptible to infectious diseases around parturition. As well, goats showed greater ChT activity in blood, colostrum, and milk than sheep. This fact may give these animals additional protection against parasite and fungal infections.

Chitotriosidase Levels in the Colostrum from Mothers of Term and Preterm Infants.

Background: Breast milk Chitotriosidase (Chit 1) shows antifungal effect and has an active role in the natural immune response against certain pathogens. The aim of this study was to compare colostrum Chit 1 levels from mothers of term and preterm infants. Materials and Methods: The study included 72 mothers of 32 preterm and 40 term infants (gestational age; 33.7 ± 1.8 vs. 39.1 ± 1.1 weeks, birth weight; 1931.7 ± 539.8 vs. 3350.9 ± 419.7 g). Breast milk samples were taken at postnatal 24-48 hours. Chit 1 level was evaluated with the quantitative calorimetric method. Results: No significant difference was determined between the term and preterm groups in terms of maternal age, education level, weight gain in pregnancy, and body mass index (BMI). The median colostrum Chit 1 level was higher in the preterm group, but the difference was not statistically significant between two groups (p = 0.43). There is no association between colostrum Chit 1 level, maternal age, gravida, BMI, infant gender, income level, and pre-eclampsia. The colostrum Chit 1 level of mothers who had weight gain exceeding the recommended limits was significantly lower than mothers with weight gain within the recommended limits in the term group (4346.2 vs. 4914.2, p = 0.021). A negative correlation was determined between the birthweight of term infants and the colostrum Chit 1 levels (p = 0.045, r = -0.319). Conclusion: Although the data need to be validated by further investigation, the observations made in this study seem to indicate that colostrum Chit-1 may have role in the protection of preterm infants.

Contrasting effects of nitrogen and phosphorus additions on soil nitrous oxide fluxes and enzyme activities in an alpine wetland of the Tibetan Plateau.

Alpine wetlands are important ecosystems, but an increased availability of soil nutrients may affect their soil nitrous oxide (N2O) fluxes and key enzyme activities. We undertook a 3-year experiment of observing nitrogen (N) and/or phosphorus (P) addition to alpine wetland soils of the Tibetan Plateau, China, with measurements made of soil extracellular enzyme activities and soil N2O fluxes. Our study showed that soil N2O flux was significantly increased by 72% and 102% following N and N+P additions, respectively. N addition significantly increased acid phosphatase (AP) and ß-1, 4-N-acetyl-glucosaminidase (NAG) activities by 32% and 26%, respectively. P addition alone exerted a neutral effect on soil AP activities, while increasing NAG activities. We inferred that microbes produce enzymes based on 'resource allocation theory', but that a series of constitutive enzymes or the treatment duration interfere with this response. Our findings suggest that N addition increases N- and P-cycling enzyme activities and soil N2O flux, whereas P addition exerts a neutral effect on P-cycling enzyme activities and N2O flux after 3 years of nutrient applications to an alpine wetland.

A high expression Mas-related G protein coupled receptor X2 cell membrane chromatography coupled with liquid chromatography and mass spectrometry method for screening potential anaphylactoid components in kudiezi injection.

Kudiezi injection is a traditional Chinese medicine injection used widely in China to alleviate blood stasis and to stimulate blood circulation. Its use has been associated with a high rate of adverse effects including anaphylactoid reactions. In the present study, a two-dimensional system comprised of high expression Mas-related G protein-coupled receptor X2 cell membrane chromatography coupled online with HPLC-ESI-IT-TOF-MS was established, and used to screen and identify anaphylactoid components in kudiezi injection. Luteolin-7-O-glucuronide, apigenin-7-O-glucronide, luteoloside and luteolin were identified as the potential anaphylactoid components. The release of ß-hexosaminidase and histamine from Laboratory of Allergic Disease 2 cells enabled evaluation of the anaphylactoid activities of these compounds in vitro. Both ß-hexosaminidase and histamine release were enhanced markedly with increasing concentrations of the anaphylactoid components. The molecular docking assay showed excellent interactions between the potential anaphylactoid constituents and MRGPRX2. In general, the two-dimensional system developed in this study is effective in screening for the anaphylactoid components in Kudiezi injection.

Anti-inflammatory effects in a mouse osteoarthritis model of a mixture of glucosamine and chitooligosaccharides produced by bi-enzyme single-step hydrolysis.

We developed a novel technique of bi-enzyme single-step hydrolysis, using recombinant chitosanase (McChoA) and exo-ß-D-glucosaminidase (AorCsxA) constructed previously in our lab, to degrade chitosan. The hydrolysis product was shown by HPLC, FTIR, and chemical analyses to be a mixture (termed "GC") composed primarily of glucosamine (80.00%) and chitooligosaccharides (9.80%). We performed experiments with a mouse osteoarthritis (OA) model to evaluate the anti-inflammatory effects of GC against OA. The three "GC groups" (which underwent knee joint damage followed by oral administration of GC at concentrations 40, 80, and 160 mg/kg·bw·d for 15 days) showed significantly downregulated serum expression of pre-inflammatory cytokines (IL-1ß, IL-6, TNF-α), and significant, dose-dependent enhancement of anti-inflammatory cytokine IL-2, in comparison with Model group. Levels of C-reactive protein, which typically rise in response to inflammatory processes, were significantly lower in the GC groups than in Model group. Thymus index and levels of immunoglobulins (IgG, IgA, IgM) were higher in the GC groups. Knee joint swelling was relieved and typical OA symptoms were partially ameliorated in the GC-treated groups. Our findings indicate that GC has strong anti-inflammatory effects and potential as a therapeutic agent against OA and other inflammatory diseases.

Combined miglustat and enzyme replacement therapy in two patients with type 1 Gaucher disease: two case reports.

BACKGROUND: Intravenous enzyme replacement therapy is a first-line therapy for Gaucher disease type 1, and substrate reduction therapy represents an oral treatment alternative. Both enzyme replacement therapy and substrate reduction therapy are generally used as monotherapies in Gaucher disease. However, one randomized study and several case reports have described combination therapy over short time periods. CASE PRESENTATION: We report two female Gaucher disease type 1 patients of mainly Anglo-Saxon descent, where combined enzyme replacement therapy and miglustat substrate reduction therapy were administered to overcome refractory clinical symptoms. The first patient was diagnosed at age 17 and developed Gaucher disease-related bone manifestations that worsened despite starting imiglucerase enzyme replacement therapy. After switching to miglustat substrate reduction therapy, her bone symptoms improved, but she developed tremors and eventually switched back to enzyme replacement therapy. Miglustat was later recommenced in combination with ongoing enzyme replacement therapy due to continued bone pain, and her bone symptoms improved along with maintained visceral manifestations. Enzyme replacement therapy was subsequently tapered off and the patient has since been successfully maintained on miglustat. The second patient was diagnosed aged 3, and commenced imiglucerase enzyme replacement therapy aged 15. After 9 years on enzyme replacement therapy she switched to miglustat substrate reduction therapy and her core symptoms were maintained/stable for 3 years. Imiglucerase enzyme replacement therapy was later added as a boost to therapy and her symptoms were subsequently maintained over a 2.3-year period. However, miglustat was discontinued due to her relocation, necessitating an increase in enzyme replacement therapy dose. Overall, both patients benefited from combination therapy. CONCLUSION: While the majority of Gaucher disease type 1 patients will not need treatment with both substrate reduction therapy and enzyme replacement therapy, the current case reports demonstrate that judicious use of combination therapy may be of benefit in some cases.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients.

BACKGROUND: Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3ß,5α,6ß-triol, both oxidized cholesterol products. OBJECTIVE: In this work, we aimed to evaluate the performance of cholestane-3ß,5α,6ß-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3ß,5α,6ß-triol value as a tool for therapy monitoring. RESULTS: Considering molecular assay as golden standard, it was verified that cholestane-3ß,5α,6ß-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3ß,5α,6ß-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. CONCLUSION: Taken together, the present data show that cholestane-3ß,5α,6ß-triol analysis has a high potential to be an important NP-C screening assay, and also can be used for therapy monitorization with miglustat in NP-C patients.

Candida albicans Impairments Induced by Peppermint and Clove Oils at Sub-Inhibitory Concentrations.

Members of Candida species cause significant health problems, inducing various types of superficial and deep-seated mycoses in humans. In order to prevent from Candida sp. development, essential oils are more and more frequently applied, due to their antifungal activity, low toxicity if used appropriately, and biodegrability. The aim of the study was to characterize the early alterations in Candida albicans metabolic properties in relation to proteins and chromosomal DNA profiles, after treatment with peppermint and clove oils at sub-inhibitory concentrations. The yeasts were affected by the oils even at a concentration of 0.0075% v/v, which resulted in changes in colony morphotypes and metabolic activities. Peppermint and clove oils at concentrations ranging from 0.015× MIC (minimal inhibitory concentration) to 0.5× MIC values substantially affected the enzymatic abilities of C. albicans, and these changes were primarily associated with the loss or decrease of activity of all 9 enzymes detected in the untreated yeast. Moreover, 29% isolates showed additional activity of N-acetyl-ß-glucosaminidase and 14% isolates-α-fucosidase in comparison to the yeast grown without essential oils addition. In response to essential oils at 0.25-0.5× MIC, extensive changes in C. albicans whole-cell protein profiles were noted. However, the yeast biochemical profiles were intact with the sole exception of the isolate treated with clove oil at 0.5× MIC. The alterations were not attributed to gross chromosomal rearrangements in C. albicans karyotype. The predominantly observed decrease in protein fractions and the yeast enzymatic activity after treatment with the oils should be considered as a phenotypic response of C. albicans to the essential oils at their sub-inhibitory concentrations and may lead to the reduction of this yeast pathogenicity.

Combination therapy in a patient with chronic neuronopathic Gaucher disease: a case report.

BACKGROUND: The variants of neuronopathic Gaucher disease may be viewed as a clinical phenotypic continuum divided into acute and chronic forms. The chronic neuronopathic form of Gaucher disease is characterized by a later onset of neurological symptoms and protracted neurological and visceral involvement. The first-choice treatment for nonneuronopathic Gaucher disease is enzyme replacement therapy with recombinant analogues of the deficient human enzyme glucocerebrosidase. Enzyme replacement therapy has been shown to improve hematological and bone manifestations associated with Gaucher disease, but, as with most proteins, recombinant enzymes cannot cross the blood-brain barrier, which prevents effects on neurological manifestations. Substrate reduction therapy with miglustat (N-butyldeoxynojirimycin) inhibits glucosylceramide synthase, which catalyzes the first step in glycosphingolipid synthesis. Because miglustat can cross the blood-brain barrier, it has been suggested that, combined with enzyme replacement therapy, it might be effective in treating neurological symptoms in patients with neuronopathic Gaucher disease. CASE PRESENTATION: We report observed effects of combined enzyme replacement therapy and substrate reduction therapy in a 7-year-old Caucasian boy with neuronopathic Gaucher disease who was homozygous for L444P mutations. He had received enzyme replacement therapy from the age of 18 months, and concomitant miglustat treatment was commenced, with dosing according to body surface area uptitrated over 1 month with dietary modifications when he reached the age of 30 months. He experienced mild diarrhea after commencing miglustat therapy, which decreased in frequency/severity over time. His splenomegaly was reduced, and his hematological values and plasma angiotensin-converting enzyme activity normalized. Plasma chitotriosidase also showed substantial and sustained decreases. After 5 years of combination therapy, the patient showed no signs of neurological impairment. CONCLUSIONS: This case supports the concept that oral miglustat in combination with intravenous enzyme replacement therapy may be beneficial in preventing neurological signs in patients with chronic neuronopathic Gaucher disease. The importance of dietary modifications has also been confirmed. Further follow-up studies are needed to better define the therapeutic effect of combined treatment in this Gaucher disease subtype.

Enhancement of Exochitinase Production by Bacillus licheniformis AT6 Strain and Improvement of N-Acetylglucosamine Production.

A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett-Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box-Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with ß-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments.

Recombinant exochitinase of the thermophilic mould Myceliopthora thermophila BJA: Characteristics and utility in generating N-acetyl glucosamine and in biocontrol of phytopathogenic fungi.

Chitinase from the thermophilic mould Myceliopthora thermophila BJA (MtChit) is an acid tolerant, thermostable and organic solvent stable biocatalyst which does not require any metal ions for its activity. To produce high enzyme titres, reduce fermentation time and overcome the need for induction, this enzyme has been heterologously expressed under GAP promoter in the GRAS yeast, Pichia pastoris. The production medium supplemented with the permeabilizing agent Tween-20 supported two-fold higher rMtChit production (5.5 × 103 U L-1 ). The consensus sequences S(132)xG(133)G(134) and D(168)xxD(171)xD(173)xE(175) in the enzyme have been found to represent the substrate binding and catalytic sites, respectively. The rMtChit, purified to homogeneity by a two-step purification strategy, is a monomeric glycoprotein of ∼48 kDa, which is optimally active at 55°C and pH 5.0. The enzyme is thermostable with t1/2 values of 113 and 48 min at 65 and 75°C, respectively. Kinetic parameters Km , Vmax , kcat , and kcat /Km of the enzyme are 4.655 mg mL-1 , 34.246 nmol mg-1  s-1 , 3.425 × 106 min-1 , and 1.36 × 10-6 mg mL-1  min-1 , respectively. rMtChit is an unique exochitinase, since its action on chitin liberates N-acetylglucosamine NAG. The enzyme inhibits the growth of phytopathogenic fungi like Fusarium oxysporum and Curvularia lunata, therefore, this finds application as biofungicide at high temperatures during summer in tropics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:70-80, 2017.

Oxidative stress, trace elements, and circulating microparticles in patients with Gaucher disease before and after enzyme replacement therapy.

UNLABELLED: We studied the level of lipid peroxide, nitric oxide (NO), trace elements (TEs), and microparticles (MPs) in Gaucher disease (GD) before and after 1 year of enzyme replacement therapy (ERT). A total of 15 children with GD and 15 healthy controls were enrolled in this study. Serum level of lipid peroxide, NO, and TEs was determined. The MPs were detected by flow cytometry. The level of lipid peroxide was significantly higher in the patients than in the controls even after ERT. Although NO level was normalized in the patients after ERT, zinc and copper were still lower in the patients after ERT. The percentages of various MPs were significantly higher in the patients than in the controls both before and after ERT. There were positive correlations between chitotriosidase and both lipid peroxide and total MPs. CONCLUSION: The GD is associated with alteration in oxidant and antioxidant status and high level of circulating MPs.

From goat colostrum to milk: physical, chemical, and immune evolution from partum to 90 days postpartum.

This study focused on the study of the changes originated in the milk from partum until d 90 of lactation. Ten multiparous Majorera goats, bred carefully under animal health standards, with a litter size of 2 kids (the average in this breed is 1.83 prolificacy) and similar gestation length (149 ± 1 d) were used. Goat kids were removed from their dams to avoid interferences with the study. Compositional content (fat, protein, and lactose) were measured, as well as some other properties, including pH, density, titratable acidity, ethanol stability, rennet clotting time, and somatic cell count. Moreover, immunity molecules (IgG, IgA, and IgM concentrations and chitotriosidase activity) received great attention. Fat and protein content were higher in the first days postpartum, whereas lactose content was lower. Density, titratable acidity, rennet clotting time, and somatic cell count decreased throughout the lactation period, whereas pH and ethanol stability increased. Relative to the immunological parameters, each measured parameter obtained its maximum level at d 0, showing the first milking as the choice to provide immunity to the newborn kids. On the other hand, this study might be used to establish what the best use is: processing or kid feeding.