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.

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.

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.

The characterization and expression analysis under stress conditions of PCST1 in Arabidopsis.

Analysis of PCST1 expression characteristics and the role of PCST1 in response to osmotic stress in Arabidopsis thaliana. The structure of PCST1 was analyzed using Bioinformatics method. Real-time PCR, GUS tissue localization and subcellular localization were adopted to analyze the expression pattern of PCST1 in Arabidopsis. To validate the transgenic positive strain of PCST1 using Real-time PCR, overexpression experiments were performed in wild type. Full-length cDNA was cloned and connected into a binary vector with 35S promoter, and the construction was transformed into wild type. With NaCl and mannitol treatments, the germination rate, green leaves rate, physiological indexes were carried out and counted in Arabidopsis with overexpression of PCST1 and T-DNA insertion mutants. The molecular mechanism of PCST1 in response to osmotic stress in Arabidopsis was analyzed. Based on the bioinformatic analysis, PCST1 is a hydrophobin with 403 amino acids, and the molecular weight is 45.3236 KDa. It contains only the START (the lipid/sterol - binding StAR - related lipid transfer protein domains) conservative domain. PCST1 possesses phosphatidylcholine binding sites and transmembrane region. Expression pattern analysis showed that expression of PCST1 increased with time. The PCST1 widely expressed in Arabidopsis, including roots, axils of stem leaves, flowers (sepal, conductive tissue of the petal, thrum, anther and stigmas), and the top and basal parts of the siliquas. It mainly localized in cell membrane. The overexpression of PCST1 enhanced the sensitivity to osmotic stress in Arabidopsis based on the germination rate. While expression of PCST1 decreased, and the sensitivity to osmotic stress had no obvious change in Arabidopsis. Its molecular mechanism study showed, that PCST1 response to osmotic stress resistance by regulating the proline, betaine synthesis, as well as the expression of key genes SOS, NCED, CIPK. PCST1 is composed of 403 amino acids. The START conservative domain, a transmembrane structure, the phosphatidyl choline binding sites are contained in PCST1. It is localized in cytoplasmic membrane. The PCST1 widely expressed in the root, leaf, flower and siliquas. NaCl and mannitol suppressed the expression of PCST1 and PCST1 can negatively control action of Arabidopsis in the osmotic stress. PCST1 regulates the synthetic pathway of proline, betaine and the expression of SOS, NCED and CIPK in response to the osmotic stress resistance.

[Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade].

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Gastroprotective effect of hydroalcoholic extract from Agaricus blazei Murill against ethanol-induced gastric ulcer in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The use of mushrooms in medicine is quite old and the first report about the use of genus Agaricus in treatment of ulcers occurred in Byzantine period. This mushroom is widely consumed as food, tea, food supplements, as well as nutraceutical and cosmeceutical applications, being cultivated and appreciated in several countries such as Brazil, Korea, Japan and China. AIM OF THE STUDY: This study aimed to characterize the chemical profile and the potential gastroprotective effect of hydroalcoholic extract from Agaricus blazei Murill (HEAb). MATERIALS AND METHODS: The extract was chemically characterized by elemental analysis, UPLC-QTOF-MSE, Nuclear Magnetic Resonance (NMR) and high-performance liquid chromatography (HPLC) techniques to elucidate the metabolites present in the extract. The quantification of phenolic compounds and the in vitro antioxidant activities were performed and the gastroprotective effect of this extract was evaluated against ethanol-induced gastric ulcer model. HEAb was administered by gavage at 5, 25 and 50 mg kg-1 and N-acetylcysteine at 300 mg kg-1 (positive control). Furthermore, the pathways of nitric oxide (NO), Cyclic Guanylate Monophosphate (cGMP), prostaglandins (PGs) and the involvement of ATP-sensitive K+ Channels were modulated. RESULTS: Mannitol, malic acid, pyroglutamic acid, L-agaritine and L-valine were putatively identified by UPLC-QTOF-MSE in HEAb. In addition, it was possible to identify mannitol by the intense signals in the NMR spectra, being still quantified as the main compound in the extract by HPLC. The contents of total phenols and flavonoids corroborated with the good antioxidant activity of HEAb. This study observed that HEAb at 25 and 50 mg kg-1 had gastroprotection effect demonstrated by the reduction of histopathological parameters and the reduction of mastocytosis in the stomach of mice. CONCLUSIONS: In this study was possible to conclude that HEAb has gastroprotective effect related to the involvement of NO and PG pathways in the ethanol-induced gastric ulcer model in mice.

Chemotherapeutics Combined with Luminal Irritants: Effects on Small-Intestinal Mannitol Permeability and Villus Length in Rats.

Chemotherapy causes intestinal mucositis, which includes villous atrophy and altered mucosal barrier function. However, there is an uncertainty regarding how the reduced small-intestinal surface area affects the mucosal permeability of the small marker probe mannitol (MW 188), and how the mucosa responds to luminal irritants after chemotherapy. The aims in this study were to determine (i) the relationship between chemotherapy-induced villus atrophy and the intestinal permeability of mannitol and (ii) how the mucosa regulate this permeability in response to luminal ethanol and sodium dodecyl sulfate (SDS). This was investigated by treating rats with a single intraperitoneal dose of doxorubicin, irinotecan, or 5-fluorouracil. After 72 h, jejunum was single-pass perfused and mannitol permeability determined at baseline and after 15 min luminal exposure to 15% ethanol or 5 mg/mL SDS. Tissue samples for morphological analyses were sampled from the perfused segment. All three chemotherapeutics caused a similar 30% reduction in villus length. Mannitol permeability increased with irinotecan (1.3-fold) and 5-fluorouracil (2.5-fold) and was reduced with doxorubicin (0.5-fold), suggesting that it is not epithelial surface area alone that regulates intestinal permeability to mannitol. There was no additional increase in mannitol permeability induced by luminal ethanol or SDS in the chemotherapy-treated rats compared to controls, which may be related to the relatively high basal permeability of mannitol compared to other common low-permeability probes. We therefore suggest that future studies should focus on elucidating the complex interplay between chemotherapy in combination with luminal irritants on the intestinal permeability of other probes.

[Effects of ferulic acid on inflammation and autophagy levels in glomerular mesangial cells induced by high glucose].

OBJECTIVE: To investigate the protective effects and possible mechanisms of ferulic acid on diabetic nephropathy by observing the effects of ferulic acid on the level of inflammation and autophagy in glomerular mesangial cells induced by high glucose. METHODS: SV40 MES 13 cells were cultured and randomly divided into the following groups: normal group (Control, 5.6 mmol/L glucose), mannitol group (Man, 30 mmol/L mannitol), high glucose group (HG, 30 mmol/L glucose), ferulic acid group (FA, 30 mmol/L glucose + 12.5, 25, 50, 100, 200 µmol/L ferulic acid), and the proliferation of SV40 MES 13 cells in each group was observed by MTT method. The levels of tumour necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and interleukin 1ß(IL-1ß)in cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of NLRP3, IL-1ß, LC3-II/I and p62 proteins in SV40 MES 13 cells were detected by Western blot. RESULTS: ①The proliferative activity of SV40 MES 13 cells was significantly higher in the HG group compared to the control group (P＜0.01), while the proliferative activity of SV40 MES 13 cells was decreased to different degrees in the FA group compared to the HG group (P＜0.05～0.01). ②Compared to the control group, the levels of TNF-α, MCP-1 and IL-1ß were increased significantly in the cell supernatant of HG group (P＜0.01). Compared with the HG group, the levels of TNF-α, MCP-1 and IL-1ß were decreased significantly in the FA group (P＜0.01). ③Compared with the control group, LC3-II/Ⅰ protein expression was decreased in the HG group, while the levels of p62, NLRP3 and IL-1ß protein were increased significantly (P＜0.01). Compared with the HG group, the expression of LC3-II/Ⅰ protein was elevated significantly (P＜0.05) in the FA group, while the levels of p62, NLRP3 and IL-1ß protein in the FA group were decreased significantly (P＜ 0.01). CONCLUSION: FA can inhibit the abnormal proliferation of SV40 MES 13 cells induced by high glucose. FA can protect glomerular mesangial cells by inhibiting inflammation and increasing the level of autophagy.

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1.

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

Targeting of GLUT5 for Transporter-Mediated Drug-Delivery Is Contingent upon Substrate Hydrophilicity.

Specific link between high fructose uptake and cancer development and progression highlighted fructose transporters as potential means to achieve GLUT-mediated discrimination between normal and cancer cells. The gained expression of fructose-specific transporter GLUT5 in various cancers offers a possibility for developing cancer-specific imaging and bioactive agents. Herein, we explore the feasibility of delivering a bioactive agent through cancer-relevant fructose-specific transporter GLUT5. We employed specific targeting of GLUT5 by 2,5-anhydro-D-mannitol and investigated several drug conjugates for their ability to induce cancer-specific cytotoxicity. The proof-of-concept analysis was carried out for conjugates of chlorambucil (CLB) in GLUT5-positive breast cancer cells and normal breast cells. The cytotoxicity of conjugates was assessed over 24 h and 48 h, and significant dependence between cancer-selectivity and conjugate size was observed. The differences were found to relate to the loss of GLUT5-mediated uptake upon increased conjugate size and hydrophobicity. The findings provide information on the substrate tolerance of GLUT5 and highlight the importance of maintaining appropriate hydrophilicity for GLUT-mediated delivery.

Mass spectrometry-based metabolomics investigation on two different indica rice grains (Oryza sativa L.) under cadmium stress.

Cadmium is a toxic environmental pollutant that is readily absorbed by rice grains and poses serious threats to human health. The selection and breeding of rice varieties with low cadmium accumulation is one of the most economical and ecological methods to reduce cadmium exposure. In this study, two different indica rice grains under cadmium stress were subjected to mass spectrometry-based metabolomics analysis for the first time. When the cadmium concentration increased in rice grains, most carbohydrates and amino acids were down-regulated, except myoinositol that can prevent cadmium toxicity, which was up-regulated. d-Mannitol and l-cysteine were up-regulated with the increase of cadmium concentration in low-cadmium-accumulating rice. Also, organic acids were activated especially 13-(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoicacid that is related to the alpha-linolenic acid metabolism and jasmonic acid production. The determination of biomarkers and characterization of metabolic pathways might be helpful for the selection of rice varieties with low cadmium accumulation.

Hsp104-dependent ability to assimilate mannitol and sorbitol conferred by a truncated Cyc8 with a C-terminal polyglutamine in Saccharomyces cerevisiae.

Tup1-Cyc8 (also known as Tup1-Ssn6) is a general transcriptional corepressor. D-Mannitol (mannitol) and D-sorbitol (sorbitol) are the major polyols in nature. Budding yeast Saccharomyces cerevisiae is unable to assimilate mannitol or sorbitol, but acquires the ability to assimilate mannitol due to a spontaneous mutation in TUP1 or CYC8. In this study, we found that spontaneous mutation of TUP1 or CYC8 also permitted assimilation of sorbitol. Some spontaneous nonsense mutations of CYC8 produced a truncated Cyc8 with a C-terminal polyglutamine. The effects were guanidine hydrochloride-sensitive and were dependent on Hsp104, but were complemented by introduction of CYC8, ruling out involvement of a prion. Assimilation of mannitol and sorbitol conferred by other mutations of TUP1 or CYC8 was guanidine hydrochloride-tolerant. It is physiologically reasonable that S. cerevisiae carries this mechanism to acquire the ability to assimilate major polyols in nature.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.

Increased Intestinal Permeability Is Associated With Later Development of Crohn's Disease.

BACKGROUND & AIMS: Increased intestinal permeability has been associated with Crohn's disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine whether increased intestinal permeability is associated with future development of CD. METHODS: We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6-35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR. RESULTS: An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64-5.63; P = 3.97 × 10-4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051-2.50; P = .029). CONCLUSIONS: Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.

Recent advances in the biotechnological production of erythritol and mannitol.

Dietary habits that include an excess of added sugars have been strongly associated with an increased risk of obesity, heart disease, diabetes, and tooth decay. With this association in view, modern food systems aim to replace added sugars with low calorie sweeteners, such as polyols. Polyols are generally not carcinogenic and do not trigger a glycemic response. Furthermore, owing to the absence of the carbonyl group, they are more stable compared to monosaccharides and do not participate in Maillard reactions. As such, since polyols are stable at high temperatures, and they do not brown or caramelize when heated. Therefore, polyols are widely used in the diets of hypocaloric and diabetic patients, as well as other specific cases where controlled caloric intake is required. In recent years, erythritol and mannitol have gained increased importance, especially in the food and pharmaceutical industries. In these areas, research efforts have been made to improve the productivity and yield of the two polyols, relying on biotechnological manufacturing methods. The present review highlights the recent advances in the biotechnological production of erythritol and mannitol and summarizes the benefits of using the two polyols in the food and pharmaceutical industries.

Import pathways of the mannityl-opines into the bacterial pathogen Agrobacterium tumefaciens: structural, affinity and in vivo approaches.

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Šresolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Šresolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.

A Sialylated-Bortezomib Prodrug Strategy Based on a Highly Expressed Selectin Target for the Treatment of Leukemia or Solid Tumors.

PURPOSE: This study aimed to explore the potential of sialic acid - related selectin targeting strategy in the treatment of leukemia and some solid tumors. We expected it could "actively" bind tumor cells and kill them, reducing non-specific toxicity to normal cells. METHODS: BOR-SA prodrug was synthesized by reacting an ortho-dihydroxy group in SA with a boronic acid group in BOR. Two kinds of leukemia cells (RAW264.7 and HL60 cells), one solid sarcoma cell model (S180 cells) and their corresponding normal cells (monocytes (MO), neutrophil (NE) and fibroblast (L929)) were selected for the in vitro cell experiments (cytotoxicity, cellular uptake, cell cycle and apoptosis experiments). The S180 tumor-bearing Kunming mice model was established for anti-tumor pharmacodynamic experiments. RESULTS: In vitro cell assay results showed that uptake of BOR-SA by HL60 and S180 cells were increased compared with the control group. BOR-SA induced a lower IC50, higher ratio of apoptosis and cell cycle arrest of tumor cells. In vivo anti-S180 tumor pharmacodynamics experiments showed that mice in the BOR-SA group had higher tumor inhibition rate, higher body weight and lower immune organ toxicity compared with the control group. CONCLUSIONS: sialic acid-mediated selectin targeting strategy may have great potential in the treatment of related tumors.

Cold Preflush of Porcine Kidney Grafts Prior to Normothermic Machine Perfusion Aggravates Ischemia Reperfusion Injury.

Normothermic machine perfusion (NMP) of kidney grafts is a promising new preservation method to improve graft quality and clinical outcome. Routinely, kidneys are washed out of blood remnants and cooled using organ preservation solutions prior to NMP. Here we assessed the effect of cold preflush compared to direct NMP. After 30 min of warm ischemia, porcine kidneys were either preflushed with cold histidine-tryptophan-ketoglutarate solution (PFNMP group) prior to NMP or directly subjected to NMP (DNMP group) using a blood/buffer solution. NMP was performed at a perfusion pressure of 75 mmHg for 6 h. Functional parameters were assessed as well as histopathological and biochemical analyses. Renal function as expressed by creatinine clearance, fractional excretion of sodium and total output of urine was inferior in PFNMP. Urine protein and neutrophil gelatinase-associated lipocalin (NGAL) concentrations as markers for kidney damage were significantly higher in the PFNMP group. Additionally, increased osmotic nephropathy was found after PFNMP. This study demonstrated that cold preflush prior to NMP aggravates ischemia reperfusion injury in comparison to direct NMP of warm ischemia-damaged kidney grafts. With increasing use of NMP systems for kidneys and other organs, further research into graft flushing during retrieval is warranted.

The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers: A Paracellular Modeling Approach.

In drug development, estimating fraction absorbed (Fa) in man for permeability-limited compounds is important but challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The Papp values of acamprosate and of several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (Pmodeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate Papp across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10-7 cm·s-1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10-7 cm·s-1), lucifer yellow (1.80 ± 0.35 × 10-7 cm·s-1), and fluorescein (2.10 ± 0.28 × 10-7 cm·s-1), and lower than that of atenolol (7.32 ± 0.60 × 10-7 cm·s-1; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the Papp of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The Pmodeled for all the paracellular markers and acamprosate was dominated by the Ppara component and matched the experimentally obtained Papp. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the Pmodeled estimated well Ppara, and the paracellular route appears to be the predominant mechanism for acamprosate Papp across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase in Staphylococcus aureus.

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.