Effects of intratesticular injection of hypertonic mannitol and saline on the quality of donkey sperm, indicators of oxidative stress and testicular tissue pathology.

OBJECTIVES: The aim of the present study was to examine donkey sperm quality after intratesticular injection of hypertonic mannitol (HM) and saline (HS). METHODS: Randomly assigned to five treatment groups were 15 adult male donkeys: (1) Control group (no treatment), (2) Surgery group (surgical castration for testosterone control), (3) NS group (normal saline intratesticular injection), (4) HS group (hypertonic saline), and (5) HM group. We injected 20 mL per testicle. We took 5 mL blood from all donkeys before injection. Castration was performed under general anesthesia 60 days later. Samples included blood and testicular tissue. Total motility (TM), progressive motility (PM), movementy features, DNA damage, morphology, viability, and plasma membrane functionality were evaluated. Hormone analyses, histomorphometric studies and oxidative stress indices including total antioxidant capacity (TAC), glutathione peroxidase (GPx), glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and NADP+/NADPH were evaluated. Apoptosis, pyroptosis-related Bax, Caspase-1, GSDMD, and Bcl-2 expression were also assessed. RESULTS: In HS and HM groups, testosterone, epididymal sperm count, motility, viability, and plasma membrane functionality dropped while sperm DNA damage increased. HS and HM groups had significantly lower histomorphometric parameters, TAC, GPx, SOD, GSH, and Bcl-2 gene expression. MDA, NADP+/NADPH, Bax, Caspase-1, and GSDMD gene expression were substantially higher in the HS and HM groups than in the control group. CONCLUSIONS: Toxic effects of hypertonic saline and mannitol on reproductive parameters were seen following, hence, they might be considered as a good chemical sterilizing treatment in donkeys.

Leuconostoc mesenteroides and Liquorilactobacillus mali strains, isolated from Algerian food products, are producers of the postbiotic compounds dextran, oligosaccharides and mannitol.

Six lactic acid bacteria (LAB) isolated from Algerian sheep's milk, traditional butter, date palm sap and barley, which produce dextran, mannitol, oligosaccharides and vitamin B2 have been characterized. They were identified as Leuconostoc mesenteroides (A4X, Z36P, B12 and O9) and Liquorilactobacillus mali (BR201 and FR123). Their exopolysaccharides synthesized from sucrose by dextransucrase (Dsr) were characterized as dextrans with (1,6)-D-glucopyranose units in the main backbone and branched at positions O-4, O-2 and/or O-3, with D-glucopyranose units in the side chain. A4X was the best dextran producer (4.5 g/L), while the other strains synthesized 2.1-2.7 g/L. Zymograms revealed that L. mali strains have a single Dsr with a molecular weight (Mw) of ~ 145 kDa, while the Lc. mesenteroides possess one or two enzymes with 170-211 kDa Mw. As far as we know, this is the first detection of L. mali Dsr. Analysis of metabolic fluxes from sucrose revealed that the six LAB produced mannitol (~ 12 g/L). The co-addition of maltose-sucrose resulted in the production of panose (up to 37.53 mM), an oligosaccharide known for its prebiotic effect. A4X, Z36P and B12 showed dextranase hydrolytic enzymatic activity and were able to produce another trisaccharide, maltotriose, which is the first instance of a dextranase activity encoded by Lc. mesenteroides strains. Furthermore, B12 and O9 grew in the absence of riboflavin (vitamin B2) and synthesized this vitamin, in a defined medium at the level of ~ 220 µg/L. Therefore, these LAB, especially Lc. mesenteroides B12, are good candidates for the development of new fermented food biofortified with functional compounds.

Direct and robust citramalate production from brown macroalgae using fast-growing Vibrio sp. dhg.

Brown macroalgae is a promising feedstock for biorefinery owing to its high biomass productivity and contents of carbohydrates such as alginate and mannitol. However, the limited availability of microbial platforms efficiently catabolizing the brown macroalgae sugars has restricted its utilization. In this study, the direct production of citramalate, an important industrial compound, was demonstrated from brown macroalgae by utilizing Vibrio sp. dhg, which has a remarkably efficient catabolism of alginate and mannitol. Specifically, citramalate synthase from Methanocaldococcus jannaschii was synthetically expressed, and competing pathways were removed to maximally redirect the carbon flux toward citramalate production. Notably, a resulting strain, VXHC, produced citramalate up to 9.8 g/L from a 20 g/L mixture of alginate and mannitol regardless of their ratios. Citramalate was robustly produced even when diverse brown macroalgae were provided directly. Collectively, this study showcased the high potential of brown macroalgae biorefinery using Vibrio sp. dhg.

Functional characterization of polyol/monosaccharide transporter 1 in Lotus japonicus.

Polyol/Monosaccharide Transporters (PLTs/PMTs) localized in the plasma membrane have previously been identified in plants. The physiological role and the functional properties of these proteins in legume plants are, however, unclear. Here we describe the functional analysis of LjPLT1, a plasma membrane-localized PLT protein from Lotus japonicus. The LjPLT1 gene was strongly expressed in the vascular tissue of roots, stems and leaves. Expression of the LjPLT1 cDNAs in yeast revealed that the protein functions as a broad-spectrum H+ -symporter for both linear polyols of sorbitol and mannitol, and cyclic polyol myo-inositol. It also catalyzes the transport of different hexoses, including fructose, glucose, galactose and mannose. Overexpression of LjPLT1 (OELjPLT1) results in inhibition of plant growth and a decrease in nodule nitrogenase activity in L. japonicus. The soluble sugars were increased in newly expanded leaves, roots and nodules but decreased in mature leaves in OELjPLT1 plants. In addition, the OELjPLT1 seedlings displayed an increased sensitivity to high content mannitol and boron toxicity, but neither drought nor salinity stresses. Taken together, the present study indicates that the LjPLT1 protein may participate in the translocation of hexoses/polyols to regulate multiple physiological and growth processes in L. japonicus.

From waste to health-supporting molecules: biosynthesis of natural products from lignin-, plastic- and seaweed-based monomers using metabolically engineered Streptomyces lividans.

BACKGROUND: Transforming waste and nonfood materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry to optimize the use of resources while reducing environmental footprint. However, despite these advancements, the production of high-value natural products often continues to depend on the use of first-generation substrates, underscoring the intricate processes and specific requirements of their biosyntheses. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce a wide array of natural products, which is attributed to its genetic versatility and potent secondary metabolic activity. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and nonfood substrates. RESULTS: We metabolically engineered S. lividans to heterologously produce the ribosomally synthesized and posttranslationally modified peptide bottromycin, as well as the polyketide pamamycin. The modified strains successfully produced these compounds using waste and nonfood model substrates such as protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. In terms of production efficiency, S. lividans showed remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, which led to enhanced and highly selective bottromycin production. Additionally, the strain generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract with no additional nutrients. CONCLUSION: Our study showcases the successful production of high-value natural products based on the use of varied waste and nonfood raw materials, circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience when grown on these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could be focused on enhancing S. lividans substrate utilization pathways to process the intricate mixtures commonly found in waste and nonfood sources more efficiently.

[Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose].

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Genome-Wide Analysis of Stress-Responsive Genes and Alternative Splice Variants in Arabidopsis Roots under Osmotic Stresses.

Plant roots show distinct gene-expression profiles from those of shoots under abiotic stress conditions. In this study, we performed mRNA sequencing (mRNA-Seq) to analyze the transcriptional profiling of Arabidopsis roots under osmotic stress conditions-high salinity (NaCl) and drought (mannitol). The roots demonstrated significantly distinct gene-expression changes from those of the aerial parts under both the NaCl and the mannitol treatment. We identified 68 closely connected transcription-factor genes involved in osmotic stress-signal transduction in roots. Well-known abscisic acid (ABA)-dependent and/or ABA-independent osmotic stress-responsive genes were not considerably upregulated in the roots compared to those in the aerial parts, indicating that the osmotic stress response in the roots may be regulated by other uncharacterized stress pathways. Moreover, we identified 26 osmotic-stress-responsive genes with distinct expressions of alternative splice variants in the roots. The quantitative reverse-transcription polymerase chain reaction further confirmed that alternative splice variants, such as those for ANNAT4, MAGL6, TRM19, and CAD9, were differentially expressed in the roots, suggesting that alternative splicing is an important regulatory mechanism in the osmotic stress response in roots. Altogether, our results suggest that tightly connected transcription-factor families, as well as alternative splicing and the resulting splice variants, are involved in the osmotic stress response in roots.

Effects of nutritional therapy on gastrointestinal microbial digestion and barrier defense markers in elderly patients with diabetes.

OBJECTIVE: We sought to investigate the effects of gastrointestinal nutrition therapy on gastrointestinal microbial digestion and barrier defense markers in elderly patients with diabetes. METHODS: A total of 120 elderly patients with type 2 diabetes were enrolled at our hospital between January 2020 and December 2022. The participants in this study were randomly allocated into either the nutritional group (n = 60) who underwent gastrointestinal nutrition therapy or the control group (n = 60) who underwent conventional T2DM diet management for a period of 12 weeks. Clinical data, as well as small intestinal permeability measured by the lactulose-mannitol urine test, plasma circulating IL-6 and zonulin levels measured by ELISA, and expressions of ZO-1 and Claudin-3 in blood analyzed through Western blotting were collected. RESULTS: The nutrition group demonstrated a higher proportion of patients achieving HbA1c < 7% compared to the control group (P < 0.05). Moreover, the nutrition group exhibited a greater reduction in fasting and postprandial blood glucose levels compared to the control group (P < 0.05). The concentrations of formate-tetrahydrofolate ligase and acetic CoA transferase were significantly increased in the nutrition group compared to the control group (P < 0.05). Fecal analysis revealed higher levels of acetic acid and butyric acid in the nutrition group compared to the control group (P < 0.05). The ratio of lactulose to mannitol was higher in the nutrition group compared to the control group (P < 0.05). Furthermore, the nutrition group showed lower levels of IL-6 and zonulin compared to the control group (P < 0.05). CONCLUSION: Personalized gastrointestinal nutrition therapy was found to enhance the production of short-chain fatty acids and preserve intestinal permeability, leading to improved gastrointestinal microbial digestion and barrier defense in elderly patients with diabetes.

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

BACKGROUND: Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. METHODS: Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. RESULTS: CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. CONCLUSIONS: Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

The transport of mannitol in Sinorhizobium meliloti is carried out by a broad-substrate polyol transporter SmoEFGK and is affected by the ability to transport and metabolize fructose.

The smo locus (sorbitol mannitol oxidation) is found on the chromosome of S. meliloti's tripartite genome. Mutations at the smo locus reduce or abolish the ability of the bacterium to grow on several carbon sources, including sorbitol, mannitol, galactitol, d-arabitol and maltitol. The contribution of the smo locus to the metabolism of these compounds has not been previously investigated. Genetic complementation of mutant strains revealed that smoS is responsible for growth on sorbitol and galactitol, while mtlK restores growth on mannitol and d-arabitol. Dehydrogenase assays demonstrate that SmoS and MtlK are NAD+-dependent dehydrogenases catalysing the oxidation of their specific substrates. Transport experiments using a radiolabeled substrate indicate that sorbitol, mannitol and d-arabitol are primarily transported into the cell by the ABC transporter encoded by smoEFGK. Additionally, it was found that a mutation in either frcK, which is found in an operon that encodes the fructose ABC transporter, or a mutation in frk, which encodes fructose kinase, leads to the induction of mannitol transport.

In-depth analysis of erythrose reductase homologs in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.

Metabolic engineering of Synechococcus elongatus for photoautotrophic production of mannitol.

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d-mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, Prbc225 , PcpcB300 , PcpcBm1 , PrbcLm17 , and PrbcLm15 . The strains were tested under the "switch conditions," where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing Prbc225 -mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD730 ) was exhibited by the engineered strain of PCC 7942 expressing PcpcB300 -mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.

The Effects of a Very-Low-Calorie Ketogenic Diet on the Intestinal Barrier Integrity and Function in Patients with Obesity: A Pilot Study.

The very-low-calorie ketogenic diet (VLCKD) is effective and safe for obese individuals, but limited information exists on its impact on the intestinal barrier. This study analyzed the effects of 8 weeks of VLCKD on 24 obese patients (11M/13F). Carbohydrate intake was fixed at 20-50 g/day, while protein and lipid intake varied from 1-1.4 g/kg of ideal body weight and 15-30 g per day, respectively. Daily calorie intake was below 800 kcal. The lactulose-mannitol absorption test assessed small intestinal permeability. Multiple markers, such as serum and fecal zonulin, fatty acid-binding protein, diamine oxidase concentrations, urinary dysbiosis markers (indican and skatole), and circulating lipopolysaccharide levels, were analyzed. Inflammation markers (serum interleukin 6, 8, 10, and tumor necrosis factor-α concentrations) were also evaluated. The results showed significant reductions in weight, BMI, and waist circumference post-diet. However, the lactulose-mannitol ratio increased by 76.5%, and a significant increase in dysbiosis markers at the end of the diet occurred. This trend was particularly evident in a subgroup of patients. Despite initial benefits, the VLCKD might negatively affect the intestinal barrier function in obese patients, potentially worsening their compromised intestinal balance.

Carrier-Mediated Process of Putrescine Elimination at the Rat Blood-Retinal Barrier.

Putrescine is a bioactive polyamine. Its retinal concentration is strictly controlled to maintain a healthy sense of vision. The present study investigated putrescine transport at the blood-retinal barrier (BRB) to gain a better understanding of the mechanisms of putrescine regulation in the retina. Our microdialysis study showed that the elimination rate constant during the terminal phase was significantly greater (1.90-fold) than that of [14C]D-mannitol, which is a bulk flow marker. The difference in the apparent elimination rate constants of [3H]putrescine and [14C]D-mannitol was significantly decreased by unlabeled putrescine and spermine, suggesting active putrescine transport from the retina to the blood across the BRB. Our study using model cell lines of the inner and outer BRB showed that [3H]putrescine transport was time-, temperature-, and concentration-dependent, suggesting the involvement of carrier-mediated processes in putrescine transport at the inner and outer BRB. [3H]Putrescine transport was significantly reduced under Na+-free, Cl--free, and K+-replacement conditions, and attenuated by polyamines or organic cations such as choline, a choline transporter-like protein (CTL) substrate. Rat CTL1 cRNA-injected oocytes exhibited marked alterations in [3H]putrescine uptake, and CTL1 knockdown significantly reduced [3H]putrescine uptake in model cell lines, suggesting the possible participation of CTL1 in putrescine transport at the BRB.

ZmCIPK32 positively regulates germination of stressed seeds via gibberellin signal.

Calcineurin B-like proteins (CBLs) as specific calcium sensors that interact with CBL-interacting protein kinases (CIPKs) play a key role in the regulation of plant development and abiotic stress tolerance. In this study, we isolated and characterized the CIPK32 gene from Zea mays. ZmCIPK32 showed that it comprised 440 amino acids and a conserved NAF motif responsible for the interaction with CBLs localized in the cytoplasm and cell membrane. The interaction of ZmCIPK32 with ZmCBL1 and ZmCBL9 demonstrated using yeast two-hybrid system and bimolecular fluorescence complementation assay required the presence of the NAF domain. Overexpression of ZmCIPK32 promoted early germination in transgenic Arabidopsis seeds relative to that observed in wild-type (WT) plants under mannitol treatment. In addition, ZmCIPK32-overexpressing plants were insensitive to treatments with exogenous abscisic acid and paclobutrazol (PBZ) at seed germination and early seedling stages. Expression levels of the key genes GA20ox and GA3ox involved in the synthesis of gibberellin (GA) were increased, whereas expression levels of genes involved in the conversion of active GA to inactive forms and GA signaling were reduced in ZmCIPK32-overexpressing plants relative to those in WT plants under mannitol and PBZ treatments. Furthermore, overexpression of ZmCIPK32 increased GA level but decreased abscisic acid level in transgenic lines compared to the respective levels in WT plants under PBZ or mannitol treatments. Our results suggest that ZmCIPK32 positively regulates seed germination under stressed conditions by modulating GA signals.

De novo biosynthesis of butyl butyrate in engineered Clostridium tyrobutyricum.

Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks.

Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives.

Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO2 employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.

Flagellin and mannitol modulate callose biosynthesis and deposition in soybean seedlings.

Callose is a polymer deposited on the cell wall and is necessary for plant growth and development. Callose is synthesized by genes from the glucan synthase-like family (GSL) and dynamically responds to various types of stress. Callose can inhibit pathogenic infection, in the case of biotic stresses, and maintain cell turgor and stiffen the plant cell wall in abiotic stresses. Here, we report the identification of 23 GSL genes (GmGSL) in the soybean genome. We performed phylogenetic analyses, gene structure prediction, duplication patterns, and expression profiles on several RNA-Seq libraries. Our analyses show that WGD/Segmental duplication contributed to expanding this gene family in soybean. Next, we analyzed the callose responses in soybean under abiotic and biotic stresses. The data show that callose is induced by both osmotic stress and flagellin 22 (flg22) and is related to the activity of ß-1,3-glucanases. By using RT-qPCR, we evaluated the expression of GSL genes during the treatment of soybean roots with mannitol and flg22. The GmGSL23 gene was upregulated in seedlings treated with osmotic stress or flg22, showing the essential role of this gene in the soybean defense response to pathogenic organisms and osmotic stress. Our results provide an important understanding of the role of callose deposition and regulation of GSL genes in response to osmotic stress and flg22 infection in soybean seedlings.

Deciphering of the Mannitol Metabolism Pathway in Clostridium tyrobutyricum ATCC 25755 by Comparative Transcriptome Analysis.

Clostridium tyrobutyricum has great potential for bio-based chemicals and biofuel production from mannitol; however, the mannitol metabolic pathway and its metabolic regulatory mechanism have not been elucidated. To this end, the RNA-seq analysis on the mid-log growth phase of C. tyrobutyricum grown on mannitol or xylose was performed. Comparative transcriptome analysis and co-transcription experiment indicated that mtlARFD, which encodes the mannitol-specific IIA component, transcription activator, mannitol-specific IIBC components, and mannitol-1-phosphate 5-dehydrogenase, respectively, formed a polycistronic operon and could be responsible for mannitol uptake and metabolism. In addition, comparative genomic analysis of the mtlARFD organization and the MtlR protein structural domain among various Firmicutes strains identified the putative cre (catabolite-responsive element) sites and conserved phosphorylation sites, but whether the expression of mannitol operon was affected by CcpA- and MtlR-mediated metabolic regulation during mixed substrate fermentation needs to be further verified experimentally. Based on the gene knockout and complementation results, the predicted mannitol operon mtlARFD was confirmed to be responsible for mannitol utilization in C. tyrobutyricum. The results of this study could be used to enhance the mannitol metabolic pathway and explore the potential metabolic regulation mechanism of mannitol during mixed substrate fermentation.

Hepatopreventive properties of hydroxytyrosol and mannitol-rich extracts obtained from exhausted olive pomace using green extraction methods.

Exhausted olive pomace (EOP) is produced in olive-pomace oil extractors as a by-product. However, the obtention of bioactive compounds from EOP can reinsert it into the economy as a new bioresource before applying other exploitation ways. The objective of the present study was to investigate the phytochemical differences between aqueous and aqueous acetonic extracts from EOP (AE-EOP and AAE-EOP, respectively) obtained by hydrothermal and ultrasound-assisted extraction, respectively. The in vitro antioxidant activities and the in vivo hepatopreventive potential were evaluated. Using RP-HPLC-ESI-QTOF-MS, the chemical profile revealed that AE-EOP and AAE-EOP showed similar qualitative profiles, with some quantitative differences. Hydroxytyrosol and mannitol were the major compounds of the extracts. The investigation of antioxidant properties in vitro highlighted that AE-EOP was slightly more efficient in scavenging DPPH, ABTS, superoxide, and hydrogen peroxide radicals, when compared to AAE-EOP. Additionally, AE-EOP and AAE-EOP showed dose-dependent suppressive effects on pancreatic lipase activity. In vivo studies showed that AE-EOP and AAE-EOP presented interesting hepatopreventive capacities against CCl4 induced liver injury, as evidenced by (i) the preventive effects against DNA damage, (ii) the normalised hepatic biomarker parameters (ALT, AST, GGT, and LDH) and (iii) the normalised lipid profile (LDL-C, TC, TG, and HDL-C) through diminishing their levels, (iv) which was supported by Oil Red O analysis. Furthermore, AE-EOP and AAE-EOP reduced the oxidative stress in liver tissue by inhibiting lipid peroxidation together with the enhancement of the hepatic antioxidant activities (CAT, SOD and GPx) and GSH content. Additionally, AE-EOP and AAE-EOP exhibited an antifibrotic effect, which was clearly demonstrated by the histopathological examination using Picrosirius red staining. The obtained results support the use of EOP extracts in industries without further purification as antioxidants and against free radical induced damage.