Identification and phenotypic characterization of patients with LADA in a population of southeast Mexico.

Latent autoimmune diabetes in adults (LADA) has clinical and metabolic features of type 1 and type 2 diabetes. LADA does not have specific features for its diagnosis apart from autoantibody detection; however, these tests are not affordable in clinical settings. In this cross-sectional study, we analyzed clinical criteria, metabolic control, pharmacological treatment, and diabetic complications in two groups of patients with diabetes -LADA and T2D- in order to identify specific characteristic of these clinical entities. Finally, we evaluated if the estimated glucose disposal rate (eGDR) and age at diagnosis of diabetes could be used as a diagnostic criterion for LADA. Demographic, biochemical, clinical and treatment were measured in 377 individuals with diabetes. The diagnostics of LADA were determined using Glutamic acid decarboxylase autoantibodies levels. Chi-square test or t-Student test were used to establish differences between groups. To identify factors associated with LADA, a logistic regression analysis was used. Finally, a ROC curve was plotted to assess the possible variables as diagnostic criteria for LADA. The 377 patients with diabetes were separated into 59 patients with LADA and 318 patients with T2D. Patients with LADA showed lower fasting glucose values, fewer diabetic complications, younger age at diagnosis of diabetes, higher insulin use, and higher eGDR in comparison to patients with T2D. Both groups had a mean BMI classified as overweight. The ROC evaluated the sensitivity and specificity, this analysis indicated that an age younger than 40.5 years and an eGDR value higher than 9.75 mg/kg/min correlated better with LADA. These parameters could be useful to identify patients suspected to have LADA at the first level of medical care in the population of southeastern Mexico and refer them to a second level of care.

Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme.

BACKGROUND: Although Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products. RESULTS: The expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h. At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level. CONCLUSION: We demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Potential applications of recombinant bifidobacterial proteins in the food industry, biomedicine, process innovation and glycobiology.

Bifidobacterial proteins have been widely studied to elucidate the metabolic mechanisms of diet adaptation and survival of Bifidobacteria, among others. The use of heterologous expression systems to obtain proteins in sufficient quantities to be characterized has been essential in these studies. L. lactis and the same Bifidobacterium as expression systems highlight ways to corroborate some of the functions attributed to these proteins. The most studied proteins are enzymes related to carbohydrate metabolism, particularly glycosidases, due to their potential application in the synthesis of neoglycoconjugates, prebiotic neooligosaccharides, and active metabolites as well as their high specificity and efficiency in processing glycoconjugates. In this review, we classified the recombinant bifidobacterial proteins reported to date whose characterization has demonstrated their usefulness or their ability to produce a product of commercial interest for the food industry, biomedicine, process innovation and glycobiology. Future directions for their study are also discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00957-1.

Analysis of formulations of probiotics accessible to the population of Tabasco, México / Análisis de formulaciones de probióticos accesibles a la población de Tabasco, México

Abstract Objective: To analyze the composition of formulations of probiotics sold in establishments in the State of Tabasco or that can be purchased online. Materials and methods: A descriptive study in which formulations of probiotics sold in 21 establishments in the city of Villahermosa were identified and compared with 30 probiotic supplements sold online. Product information was organized in a database and analyzed according to the dosage form, probiotic genera, species/subspecies contained and their classification as probiotics or synbiotics and as drugs or supplements. Results: Thirty-one local products and 30 online products formulated with probiotics in 6 different dosage forms were analyzed. Only five local products and no online products are certified by COFEPRIS as drugs. Forty-eight percent of the formulations are monostrain and the rest are multistrain. Seventy-two percent of the formulations are probiotics and the rest are synbiotics. Among the 61 products analyzed, 46 species belonging to 13 different genera were identified, and 39% were common to local and online products. Many of products contain species of the Lactobacillus and Bifidobacterium genera. The genus Bacillus was the only genus that was never combined with other genera in the formulations analyzed in this study. Conclusions: The population of the state of Tabasco can find at least 31 formulations of probiotics in local establishments. The variety increases if we consider the dietary supplements available for sale online. Multistrain supplements are particularly abundant in online retailers. The products certified by COFEPRIS ensure that the benefits of the formulation are supported by clinical trials in humans and are manufactured following good manufacturing practices.

Resumen Objetivo: Analizar la composición de formulaciones de probióticos que se venden en establecimientos del Estado de Tabasco o que se pueden comprar en línea. Materiales y métodos: Estudio descriptivo en el que se identificaron formulaciones de probióticos comercializados en 21 establecimientos de la ciudad de Villahermosa y se compararon con 30 suplementos probióticos comercializados en línea. La información del producto se organizó en una base de datos y se analizó de acuerdo con la forma de dosificación, los géneros y especies / subespecies de probióticos presentes y su clasificación como probióticos o simbióticos y como medicamentos o suplementos. Resultados: Se analizaron 31 productos locales y 30 productos de venta en línea formulados con probióticos en 6 formas de dosificación diferentes. Solo cinco productos locales y ningún producto en línea están certificados por COFEPRIS como medicamentos. El 48% de las formulaciones son monocepa y el resto son multicepa. El 72% de las formulaciones son probióticos y el resto son simbióticos. Entre los 61 productos analizados, se identificaron 46 especies pertenecientes a 13 géneros diferentes y el 39% fueron comunes a productos locales y de venta en línea. Muchos de los productos contienen especies de los géneros Lactobacillus y Bifidobacterium. El género Bacillus fue el único que nunca se combinó con otros géneros en las formulaciones analizadas en este estudio. Conclusiones: La población del estado de Tabasco puede encontrar al menos 31 formulaciones de probióticos en establecimientos locales. La variedad aumenta si tenemos en cuenta los suplementos dietéticos disponibles de venta en línea. Los suplementos multicepa son particularmente abundantes en los productos en línea. Los productos certificados por COFEPRIS aseguran que los beneficios de la formulación están respaldados por ensayos clínicos en humanos y se fabrican siguiendo buenas prácticas de fabricación.

Trypsin gene expression in adults and larvae of tropical gar Atractosteus tropicus.

Trypsin gene (try) expression levels were quantified in different organs of wild and captive tropical gar (Atractosteus tropicus) adults, and changes in expression during initial ontogeny of the species were determined. RNA was extracted from the pancreas, and cDNA was synthesized and later amplified by endpoint PCR using oligonucleotides designed from different try sequences of fish registered in GenBank. Subsequently, specific oligonucleotides were designed from the partial sequences. Gene expression was measured after RNA extraction and synthesis of the cDNA of 11 organs (liver, pancreas, stomach, esophagus, intestine, pyloric caeca, brain, muscle, gills, gonad, and kidney) of captive and wild adults. Likewise, samples of A. tropicus larvae were taken on days 0 (embryo), 5, 10, 15, 20, 25, and 30 days after hatching (DAH), the RNA was extracted, and the synthesis of cDNA was carried out to measure real-time gene expression (qPCR). The results showed that the highest relative try expression occurred mainly in the esophagus, liver, stomach, and pancreas of both wild and captive adult fish; however, captive organisms had a higher try expression level than wild fish. Although try expression during initial ontogeny was high in embryos (0 DAH), it did not reach the maximum value until 15 DAH. It was concluded that try expression levels in captive adults are due to the high protein content in the balanced feed (trout diet). The highest try expression level during larviculture was detected at 15 DAH, which indicates that A. tropicus larvae have a mature digestive system and can efficiently hydrolyze proteins from feed at this developmental stage.

Fish trypsins: potential applications in biomedicine and prospects for production.

In fishes, trypsins are adapted to different environmental conditions, and the biochemical and kinetic properties of a broad variety of native isoforms have been studied. Proteolytic enzymes remain in high demand in the detergent, food, and feed industries; however, our analysis of the literature showed that, in the last decade, some fish trypsins have been studied for the synthesis of industrial peptides and for specific biomedical uses as antipathogenic agents against viruses and bacteria, which have been recently patented. In addition, innovative strategies of trypsin administration have been studied to ensure that trypsins retain their properties until they exert their action. Biomedical uses require the production of high-quality enzymes. In this context, the production of recombinant trypsins is an alternative. For this purpose, E. coli-based systems have been tested for the production of fish trypsins; however, P. pastoris-based systems also seem to show great potential in the production of fish trypsins with higher production quality. On the other hand, there is a lack of information regarding the specific structures, biochemical and kinetic properties, and characteristics of trypsins produced using heterologous systems. This review describes the potential uses of fish trypsins in biomedicine and the enzymatic and structural properties of native and recombinant fish trypsins obtained to date, outlining some prospects for their study.

Levan-type fructooligosaccharides synthesis by a levansucrase-endolevanase fusion enzyme (LevB1SacB).

We describe here the enzymatic production of levan type-fructooligosaccharides (L-FOS) with a DP from 2 to 10, through simultaneous synthesis and hydrolysis reactions. This was accomplished by LevB1SacB, a new enzyme resulting from the fusion of SacB, a levansucrase from Bacillus subtilis and LevB1, an endolevanase from B. licheniformis. In the fusion enzyme, SacB retains its catalytic behavior with a decrease in kcat from 164 to 108s-1. LevB1 in LevB1SacB kinetic behavior improves considerably reaching saturation with levan and following Michaelis-Menten kinetics, quite differently from the previously reported first order kinetic behavior. We also report that LevB1SacB or both enzymes (LevB1 & SacB) at equimolar concentrations in simultaneous reactions result in an optimal, wide and diverse L-FOS profile, including 6-kestose, levanbiose and blastose among other L-FOS and 1-kestose, which accumulates as by-product of SacB levan synthesis. Yields of around 40% (w/w) were obtained from 600g/l sucrose with either LevB1SacB or LevB1 & SacB. The reaction was successfully scaled up to a stirred 2l bioreactor.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

Association between Polymorphisms of the DRD2 and ANKK1 Genes and Suicide Attempt: A Preliminary Case-Control Study in a Mexican Population.

BACKGROUND: The aim of this study was to analyze the possible association of polymorphic variants of the DRD2 and ANKK1 genes with suicide attempt in a Mexican population. METHODS: We conducted a case-control study in 289 subjects (166 suicide attempters and 123 healthy controls). We genotyped 2 polymorphisms of DRD2 (rs6275 and rs1799978) and 1 polymorphism of ANKK1 (rs1800497); then we analyzed the association between suicide attempt and these polymorphisms through genotypes, alleles, and inheritance models. RESULTS: Individuals who carried the TT genotype of the rs1800497 showed a 3-fold risk of attempting suicide (OR = 3.01; 95% CI 1.56-5.81, p = 0.001) when evaluated through the recessive model. In an analysis stratified by gender, this risk factor remained present among females (OR = 2.81; 95% CI 1.37-5.75) as well as males (OR = 3.3; 95% CI 1.01-10.77). CONCLUSION: Our results suggest that the rs1800497 variant of the ANKK1 gene could increase the risk of suicide attempt in a Mexican population. However, further studies using larger samples are necessary to obtain more conclusive results.

Bacillus subtilis 168 levansucrase (SacB) activity affects average levan molecular weight.

Levan is a fructan polymer that offers a variety of applications in the chemical, health, cosmetic and food industries. Most of the levan applications depend on levan molecular weight, which in turn depends on the source of the synthesizing enzyme and/or on reaction conditions. Here we demonstrate that in the particular case of levansucrase from Bacillus subtilis 168, enzyme concentration is also a factor defining the molecular weight levan distribution. While a bimodal distribution has been reported at the usual enzyme concentrations (1 U/ml equivalent to 0.1 µM levansucrase) we found that a low molecular weight normal distribution is solely obtained al high enzyme concentrations (>5 U/ml equivalent to 0.5 µM levansucrase) while a high normal molecular weight distribution is synthesized at low enzyme doses (0.1 U/ml equivalent to 0.01 µM of levansucrase).

Production of functional oligosaccharides through limited acid hydrolysis of agave fructans.

A controlled acid thermal hydrolysis process of fructans from agave (Agave tequilana Weber var. azul) was designed to produce a mixture of functional prebiotic fructooligosaccharides and sweetening power. Despite its highly branched structure, first-order kinetic behaviour with respect to substrate concentration was found with an activation energy of 95kJ/mol, similar to the value found for other linear fructans such as chicory inulin. Fructose equivalent (FE) an analogous parameter to dextrose equivalent (DE) used in the starch industry was introduced to characterise fructan hydrolysis; maximum oligosaccharide production was observed when fructose equivalent (FE) reached 27-48 with a structural profile analysed by high-performance anion-exchange chromatography HPAEC-PAD. After hydrolysis, glucose and fructose may be eliminated through a biological purification step involving the addition of Pichia pastoris cells, which selectively consume these sugars but are unable to metabolise fructooligosaccharides.

Enzymatic hydrolysis of fructans in the tequila production process.

In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.