Total Adenosine Research Articles

Adenosine A2A Receptors in Bone Marrow-Derived Cells Attenuate Cognitive Impairment in Mice After Chronic Hypoperfusion White Matter Injury

The mechanism of cognitive dysfunction caused by ischemic white matter lesions is unclear. To explore the effect and mechanism of different cell-derived adenosine A2A receptor (A2AR) in cognitive impairment caused by chronic hypoperfusion white matter lesions (CHWMLs), we destroyed the bone marrow hematopoietic capacity of the recipient mice using radiation irradiation followed by establishing the selectively inactivated or reconstituted A2AR models with the transplanting bone marrow from global A2AR gene knockout or wild-type mice into wild-type or gene knockout mice, respectively. Then Morris Water Maze (MWM), ELISA, immunohistochemistry, and Bielschowsky silver staining were used to assess the effect and mechanism of the cognitive function in chronic cerebral blood flow hypoperfusion (CCH) model. Selectively reconstructing bone marrow-derived cells (BMDCs) A2AR (WT → KO group) and activated total adenosine A2AR with CGS21680 (CCH + CGS group) improved the cognitive related index. Activation of BMDC A2AR suppressed expression of inflammatory cytokines in peripheral blood and reduced the number of activated microglia cells co-localized with cystatin F in local brain, consequently inhibited white matter lesions. On the contrary, selective inactivation of adenosine A2AR (KO → WT group) and activation of non-BMDC A2AR with CGS21680 (KO → WT + CGS group) served the opposite effects. These results suggested that BMDC A2AR could inhibit white matter lesions and attenuate cognitive impairment after CHWMLs, whereas non-BMDC A2ARs aggravate cognitive impairment. The systemic inflammatory response and local activated microglia with cystatin F high expression were involved in the process of cognitive function recovery with BMDC A2AR. The overall trend is that BMDC A2ARs play a leading role.

A comparison between chemical cleaning efficiency in lab-scale and full-scale reverse osmosis membranes: Role of extracellular polymeric substances (EPS)

Chemical cleaning is vital for the optimal operation of membrane systems. Membrane chemical cleaning protocols are often developed in the laboratory flow cells (e.g., Membrane Fouling Simulator (MFS)) using synthetic feed water (nutrient excess) and short experimental time of typically days. However, full-scale Reverse Osmosis (RO) membranes are usually fed with nutrient limited feed water (due to extensive pre-treatment) and operated for a long-time of typically years. These operational differences lead to significant differences in the efficiency of chemical Cleaning-In-Place (CIP) carried out on laboratory-scale and on full-scale RO systems. Therefore, we investigated the suitability of lab-scale CIP results for full-scale applications. A lab-scale flow cell (i.e., MFSs) and two full-scale RO modules were analysed to compare CIP efficiency in terms of water flux recovery and biofouling properties (biomass content, Extracellular Polymeric Substances (EPS) composition and EPS adherence) under typical lab-scale and full-scale conditions. We observed a significant difference between the CIP efficiency in lab-scale (~50%) and full-scale (9–20%) RO membranes. Typical biomass analysis such as Total Organic Carbon (TOC) and Adenosine triphosphate (ATP) measurements did not indicate any correlation to the observed trend in the CIP efficiency in the lab-scale and full-scale RO membranes. However, the biofilms formed in the lab-scale contains different EPS than the biofilms in the full-scale RO modules. The biofilms in the lab-scale MFS have polysaccharide-rich EPS (Protein/Polysaccharide ratio = 0.5) as opposed to biofilm developed in full-scale modules which contain protein-rich EPS (Protein/Polysaccharide ratio = 2.2). Moreover, EPS analysis indicates the EPS extracted from full-scale biofilms have a higher affinity and rigidity to the membrane surface compared to EPS from lab-scale biofilm. Thus, we propose that CIP protocols should be optimized in long-term experiments using the realistic feed water.

Effects of fasting on isolated murine skeletal muscle contractile function during acute hypoxia.

Stored muscle carbohydrate supply and energetic efficiency constrain muscle functional capacity during exercise and are influenced by common physiological variables (e.g. age, diet, and physical activity level). Whether these constraints affect overall functional capacity or the timing of muscle energetic failure during acute hypoxia is not known. We interrogated skeletal muscle contractile properties in two anatomically distinct rodent hindlimb muscles that have well characterized differences in energetic efficiency (locomotory- extensor digitorum longus (EDL) and postural- soleus muscles) following a 24 hour fasting period that resulted in substantially reduced muscle carbohydrate supply. 180 mins of acute hypoxia resulted in complete energetic failure in all muscles tested, indicated by: loss of force production, substantial reductions in total adenosine nucleotide pool intermediates, and increased adenosine nucleotide degradation product-inosine monophosphate (IMP). These changes occurred in the absence of apparent myofiber structural damage assessed histologically by both transverse section and whole mount. Fasting and the associated reduction of the available intracellular carbohydrate pool (~50% decrease in skeletal muscle) did not significantly alter the timing to muscle functional impairment or affect the overall force/work capacities of either muscle type. Fasting resulted in greater passive tension development in both muscle types, which may have implications for the design of pre-clinical studies involving optimal timing of reperfusion or administration of precision therapeutics.

Impact of backwash on biofiltration-related nitrogenous disinfection by-product formation

Previous studies have reported that biofilm extracted from full-scale biofilters can serve as nitrogenous disinfection by-product (N-DBP) precursors. Detached biofilm materials could escape during filter ripening and form N-DBP upon chloramination. This study examined the potential breakthrough of biofilm and N-DBP precursors during filter ripening at two water treatment plants (WTPs). The presence of biofilm material in aqueous samples was estimated by total adenosine triphosphate (tATP) levels; N-DBP formation potential (FP) tests were conducted under uniform formation conditions to quantify N-nitrosodimethylamine (NDMA) and haloacetonitrile (HAN4) precursors. While tATP peaks in filter effluent were observed post backwash at both WTPs, temporary increases of effluent NDMA FP were only observed during filter ripening where particle-associated NDMA precursors served as the dominant contributor. Overall, biofilters examined in this study demonstrated a consistent removal of NDMA FP regardless of the filter ripening process.

Diagnostic Value of Serum Adenosine Deaminase and Its Isoenzymes for Autoimmune Liver Disease

Background: Adenosine Deaminase (ADA) has been found to be involved in autoimmune disease progression. Objectives: We aimed to evaluate the diagnostic value of serum ADA activity in Autoimmune Liver Disease (AILD). Methods: The study included 50 AILD patients, 33 viral hepatitis patients, and 60 healthy subjects. The serum levels of total Adenosine Deaminase (tADA) and its isoenzymes (ADA1 and ADA2) were determined. The Receiver Operating Characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of serum ADA activity. Results: Our results showed that the serum tADA and ADA2 levels were significantly higher in AILD patients (tADA: 19 (IQR 14 - 24) U/L; ADA2: 16 (IQR 10 - 19) U/L) than in healthy controls (tADA: 9 (IQR 7 - 11) U/L; ADA2: 7 (IQR 6 - 9) U/L), while there was no significant difference in serum ADA1 activity between AILD and healthy subjects. Based on the ROC curves analysis, the optimal cutoff values of serum tADA and ADA2 activity were 11.5 and 9.5 U/L, respectively. At this level, the highest diagnostic values of tADA (specificity: 83.3%; sensitivity: 88%) and ADA2 (specificity: 85.0%; sensitivity: 82%) were obtained. Moreover, our results showed no significant difference in serum tADA, ADA1, and ADA2 levels between AILD and viral hepatitis patients (P = 0.049; P = 0.29; P = 0.054). Conclusions: Serum ADA activity can be used to distinguish AILD patients from healthy subjects, but it cannot be used in the differential diagnosis of AILD and viral patients.

Analytical characterization of trace elements (zinc, copper, cadmium, lead and selenium) in saliva of pigs under common pathological conditions in the field: a pilot study

BackgroundThis study is focused on the measurement of trace elements (zinc, copper, cadmium, lead and selenium) in the saliva of pigs in order to study their levels on different porcine pathological conditions in the field. The experiment involved 15 pigs without clinical signs of disease and 42 diseased pigs (suffering from lameness, rectal prolapse, fatigue or growth rate retardation). Individual saliva samples were collected, allowing the pigs to chew a sponge each for trace element quantifications through atomic absorption spectrometry (AAS). Since this is the first report on the measurements of trace elements in porcine saliva, a routine analytical validation study was performed for the quantification of all the studied elements. Moreover, the acute phase proteins C-reactive protein (CRP) and haptoblobin (Hp), the total antioxidant capacity (TAC) and adenosine deaminase (ADA) were quantified in the saliva samples for the animal’s health status assessment.ResultsModifications in the levels of acute phase proteins or ADA were only recorded in animals with lameness and rectal prolapse and those with fatigue respectively. Moreover, TAC level changes were observed in pigs with growth-rate retardation. However, alterations in the levels of two or more trace elements were reported for all the different groups of diseased pigs with evident variations within pathologies.ConclusionsThe salivary quantification of trace elements could be considered as a complementary tool to acute phase proteins, TAC and ADA determinations for disease detection and differentiation in the pig and should be explored in greater depth.

Influence of the electret electric field on osteoreparation and inflammation in surgical treatment of simulated tuberculous osteitis estimated by morphological and biochemical criteria

Introduction. Recent trends in orthopedic surgery target factors and conditions that affect the destructive process-es in bone tissue and the effectiveness of reconstructive surgery. Electrets based on anodic tantalum oxide with negative external surface charge (hereinafter, “electret”) create a quasi-static long-term electric field in the sur-rounding space. Their implantation in the surgical treat-ment of early stages of degenerative joint diseases is accompanied by pronounced analgesic effect and signi-ficant increase in the quality of life of patients. Objective.To identify the features of osteogenesis and regression of specific inflammation in the surgical treatment of ex-perimental tuberculous osteitis in a constant electric field created by electret. Materials and methods. We studied the processes of osteoreparation and dynamics of the bone tissue specific inflammation activity in 30 rabbits with simulated tuberculous osteitis of the distal (meta-diaphysis) femur. 3 animals were intact (group 1), 6 rab-bits did not receive any therapy after tuberculous oste-itis simulation (group 2), 9 rabbits (group 3) underwent necrectomy and osteoplasty with autograft, 9 rabbits (group 4) underwent necrectomy and electret implanta-tion (with further generation of quasi-static electric field), and 6 animals (group 5) underwent necrectomy, osteo-plasty with autograft and perifocal electret implantation. Animals in groups 3, 4 and 5 received antituberculous therapy. The observation period was 1, 2 and 4 months after the grafting procedure. Bone regeneration and in-flammation regression were assessed with morphologi-cal and biochemical methods (enzymes of the purinergic system — activity of total adenosine deaminase and its isoenzymes, elastase activity, level of acute phase reac-tants: ceruloplasmin, haptoglobin, α1-acid glycoprotein, and albumin). Results. The evolution of tuberculous in-flammation of bone tissue was observed for the control periods in animals with simulated tuberculous osteitis. The analysis of bone tissue reparation and inflammation activity regression showed that only a combination of antituberculosis therapy, necrectomy, osteoplasty with autograft and electret perifocal implantation allowed in 2 months to achieve complete regression of active tuber-culosis and replacement of defect with mature trabec-ular bone. Conclusion. The electret electric field based on anodic tantalum oxide with negative external surface charge accelerates the processes of osteoreparation and reduces the activity of tuberculous inflammation in the surgical treatment of experimental tuberculous osteitis.

Substantially enhanced anaerobic reduction of nitrobenzene by biochar stabilized sulfide-modified nanoscale zero-valent iron: Process and mechanisms

Nanoscale zero-valent iron (nZVI), although being increasingly used in anaerobic systems for strengthening the removal of various refractory pollutants, is limited by various inherent drawbacks, such as easy precipitation, passivation, poor mass and electron transfer. To address the above issues, biochar stabilized sulfide-modified nZVI (S-nZVI@BC) was added into an up-flow anaerobic sludge blanket (UASB) to investigate the enhancement of anaerobic biodegradation of nitrobenzene (NB) and its impacts on microbial community structure. The results demonstrated that both NB reduction and aniline formation could be substantially facilitated in S-nZVI@BC coupled system compared to other anaerobic ones coupled with nZVI or S-nZVI. The dosage of S-nZVI@BC resulted in the formation of densely packed aggregates, evidently increased the extracellular polymeric substances content, promoted the volatile fatty acids transformation and stimulated the methane yield. Furthermore, species related to fermentation (Bacteroides and Longilinea), methanogenesis (Methanosarcina and Methanomethylovorans), electroactivity (Pelobacter, Thiobacillus and Phaselicystis) as well as reduction (Desulfovibrio) were considerably enriched in S-nZVI@BC coupled system. The activities of electron transport, total adenosine triphosphate, nitroreductase and NAD(P)H, which were closely related to microbial activity and NB transformation, were increased noticeably in S-nZVI@BC coupled anaerobic system. This study demonstrated the promising potential for long-term operation and full-scale application of S-nZVI@BC coupled system for the treatment of NB containing wastewater.

Nano selenium treatment effects on thyroid hormones, immunity and antioxidant status in rabbits

<p>The present study was conducted to compare the effect of Nano-Selenium (Nano-Se) and sodium selenite (SSe) on antioxidant enzyme activity, immunity and thyroid activity of growing New Zealand White (NZW) rabbits. In this study, 72 male rabbits (5 wk old) were divided randomly into 3 groups (24 rabbits each). The first group served as a placebo; in groups 2 and 3, each rabbit was intramuscularly injected once a week with 4 mL solution of Nano-Se or SSe, respectively, for a 2-mo period. The solution was adjusted to provide 30 μg Se/kg/live body weight. Results showed that Nano-Se treatment significantly (<em>P</em><0.0001) increased in superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione (GSH) more than control, but decreased significantly each of glutathione disulphide (GSSG) and nitric oxide (NO) levels in serum. Likewise, supplementation of SSe increased (<em>P</em><0.0001) GPx activity and significantly decreased both malondialdehyde (MDA) and GSSG levels more than control. Nano-Se significantly enhanced serum IgG and IgM more than SSe and control groups. Serum aspartate aminotransferase increased (<em>P</em><0.0001) due to Nano-Se treatment as compared to control and SSe, although the lowest (<em>P</em><0.0001) activity of alanine aminotransferase was recorded due to SSe supplementation. Nano-Se treatment increased (<em>P</em><0.0001) both T3 and T4 concentrations more than other groups. Furthermore, administration of Nano-Se increased SOD, GPx, GSH, total antioxidant capacity (TAC) and adenosine triphosphate (ATP) in liver tissue of growing rabbits, while it decreased MDA and 8-hydroxy-2’deoxyguanosine (8-oHdG) levels in liver tissue compared with control. Also, SSe showed an increase (<em>P</em><0.0001) in GSH, and ATP, but significantly decreased TAC and MDA levels compared with control. It can be concluded that Nano-Se supplementation significantly enhanced the activity of antioxidant enzymes in both serum and liver tissues, with a greater positive influence on immunoglobulin production and thyroid activity in growing NZW rabbits than SSe.</p>

Potential of hydrocarbon and oxygenated monoterpenes against Culex pipiens larvae: Toxicity, biochemical, pharmacophore modeling and molecular docking studies

Culex pipiens is a main vector for Bancroftian filariasis, Rift Valley Fever and diseases caused by other viruses, leaving several peoples with disabilities. In recent years, plant derived compounds have received much attention as potential alternatives to synthetic chemicals due to their low toxicity to mammals and environmental persistence. Twenty-one monoterpenes from different chemical groups (hydrocarbons and oxygenated products) were evaluated against Culex pipiens larvae. In addition, in vivo biochemical studies including effects on acetylcholine esterase (AChE), acid and alkaline phosphatases (ACP and ALP), total adenosine triphosphatase (ATPase) and gamma-aminobutyric acid transaminase (GABA-T) were investigated. Furthermore, in silico studies including pharmacophore elucidation, ADMET analysis and molecular docking of these compounds were performed. Among all tested monoterpenes, hydrocarbons [p-cymene, (R)-(+)-limonene and (+)-α-pinene], acetates (cinnamyl acetate, citronellyl acetate, eugenyl acetate and terpinyl acetate), alcohols [(±)-β-citronellol and terpineol], aldehydes [citral and (1R)-(−)-myrtenal] and ketone [(R)-(+)-pulegone] exhibited the highest larval toxicity with LC50 = 14.88, 27.97, 26.13, 2.62, 3.81, 2.74, 21.65, 1.64, 21.70, 21.76, 1.68 and 1.90 mg/L after 48 h of exposure, respectively. The compounds proved a significant inhibition of all tested enzymes except total ATPase. The biochemical and molecular docking studies proved that AChE and GABA-T were the main targets for the tested monoterpenes.

An Improved Specimen Handling Procedure for Pathogen Detection of the Cerebrospinal Fluid by Microscope.

In order to increase the detection rate of pathogenic microorganisms in CSF, an improved specimen handling procedure (ISHP) was created. This study enrolled encephalitis and control groups, both groups were handled with traditional specimen handling procedure (TSHP) and ISHP. Glutaraldehyde was added to the ISHP. Observed items included: total protein, glucose, chloride, adenosine deaminase, lactate dehydrogenase, sediment, cell and pathogen, Pandy's test. Sediment test of CSF: There was 1 specimen in 10 control specimens tested by TSHP in which Pandy's test was positive; there were 2 specimens tested by ISHP which could see sediment by eye. There was no statistical difference between those two methods (p = 1.000, Table 1). Ten specimens in 23 of the encephalitis group processed by TSHP were positive with Pandy's test; 23 specimens processed by ISHP could all see sediments by eye (Figure 1). There was a statistical difference between the two methods (p = 0.000, Table 1). Pathogen test of CSF: no pathogen was found in the control group processed by TSHP and ISHP. No pathogen was found in the encephalitis group specimens processed by TSHP. Pathogen tests were positive in 7 encephalitis specimens processed by ISHP (p = 0.009, Table 1), which were confirmed as Rickettsia spp. by Gimenze stain (Figure 1B), IFA (Figure 2). The results revealed that ISHP contributes to the separation of cells, pathogens (such as Rickettsia), and proteins.

Involvement of the phosphoryl transfer network on cardiac energetic metabolism during Staphylococcus aureus infection and its association to disease pathophysiology

Evidences have suggested that the phosphoryl transfer network by the enzymatic activities of creatine kinase (CK), adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), shows new perspectives to understand some disturbances in the energy metabolism during bacterial infections. Thus, the aim of this study was to evaluate whether Staphylococcus aureus infection in mice could alter serum and cardiac activities of these enzymes and their association to disease pathophysiology. For that, we measured total leukocytes, lymphocytes and neutrophils (just 48 h of infection) that were lower in infected animals after 48 and 72 h in infected mice compared with negative control, while total protein and globulin plasma levels were higher after 72 h of infection. The serum CK activity was higher in infected animals 48 and 72 h post-infection compared to the control group, as well as observed for mitochondrial cardiac CK activity. The serum PK activity was higher in infected animals after 72 h of infection compared to the control group, and lower in the cardiac tissue. The cardiac AK activity was lower in infected animals 48 h and 72 h post-infection compared to the control group, while serum and cardiac LDH activities were higher. Based on these evidences, it is possible to conclude that the stimulation of CK activity exerts a key role as an attempt to maintain the bioenergetic homeostasis by the production of phosphocreatine to avoid a rapid fall on the concentrations of total adenosine triphosphate. In summary, the phosphoryl transfer network can be considered a pathway involved in the improvement on tissue and cellular energy homeostasis of S. aureus-infected mice.

Early embryonic exposure of ionizing radiations disrupts zebrafish pigmentation.

Studies have demonstrated that zebrafish are powerful tools for monitoring environmental toxicity, including radiation hazard. Here we investigated the developmental toxicity of ionizing radiation (IR) in an in vivo embryonic zebrafish model. The effects of heavy ion (12 C6+ ), proton, and X-ray radiation on early zebrafish embryos were determined. A similar dose-dependent decrease in the hatch and survival rate of zebrafish embryos was observed after exposure to these irradiations. Exposure of zebrafish embryos to 1-4 Gy IR caused significant loss of pigmentation. Quantitative real-time reverse transcription polymerase chain reaction, western blot analysis, and in situ hybridization (ISH) experiment revealed that atp5α1 was markedly upregulated in irradiated zebrafish embryos. In addition, IR resulted in a rapid decrease in total adenosine triphosphate (ATP) generation. With dual functions of synthesizing or hydrolyzing ATP, ATP synthase regulated H+ transport crossing the mitochondrial inner. Administration of the mitochondrial ATP synthase inhibitor, oligomycin, partially restored pigmentation in irradiated zebrafish embryos, but the ATPase inhibitor, BTB06584, had no effect. Taken together, these results showed that IR exposure downregulated zebrafish pigmentation through regulation of H+ ion transport in mitochondria.

Inactivation of two Mycobacteria by free chlorine: Effectiveness, influencing factors, and mechanisms

Chlorination is one of the most widely used disinfection techniques, and the problem of “chlorine-resistant bacteria” (CRB) has attracted more attention recently. In this study, the deactivation of typical CRB in water, Mycobacterium fortuitum (M. fortuitum) and Mycobacterium mucogenicum (M. mucogenicum), by free chlorine was investigated with Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) as the reference. The chlorination effectiveness of chlorine on M. fortuitum and M. mucogenicum and the effect of chlorine concentration, pH, and humic acid were studied. It was found that M. mucogenicum was more resistant to chlorine than M. fortuitum, both of which were much more resistant than E. coli and B. subtilis. The effect of disinfectant concentration on the inactivation efficiency was positive, whereas the influence of pH and humic acid was negative. The inactivation mechanisms were explored by analyzing the bacteria morphology, the destruction of cell membrane, the cell hydrophobicity, as well as total adenosine triphosphate (ATP), superoxide dismutase (SOD) activity. The slight destruction of the cell membrane was observed after deactivation with chlorine, and high hydrophobicity of the cell membrane combined with metabolic changes might lead to the chlorine tolerance of Mycobacteria.

The role of salinity on the changes of the biomass characteristics and on the performance of an OMBR treating tannery wastewater

Tannery wastewaters are difficult to treat biologically due to the high salinity and organic matter concentration. Conventional treatments, like sequential batch reactors (SBR) and membrane bioreactors (MBR), have showed settling problems, in the case of SBR, and ultrafiltration (UF) membrane fouling in the case of MBR, slowing their industrial application. In this work, the treatment of tannery wastewater with an osmotic membrane bioreactor (OMBR) is assessed. Forward osmosis (FO) membranes are characterized by a much lower fouling degree than UF membranes. The permeate passes through the membrane pores (practically only water by the high membrane rejection) from the feed solution to the draw solution, which is also an industrial wastewater (ammonia absorption effluent) in this work. Experiments were carried out at laboratory scale with a FO CTA-NW membrane from Hydration Technology Innovations (HTI). Tannery wastewater was treated by means of an OMBR using as DS an actual industrial wastewater mainly consisting of ammonium sulphate. The monitoring of the biological process was carried out with biological indicators like microbial hydrolytic enzymatic activities, dissolved and total adenosine triphosphate (ATP) in the mixed liquor and microbial population. Results indicated a limiting conductivity in the reactor of 35 mS cm−1 (on the 43th operation day), from which process was deteriorated. This process performance diminution was associated by a high decrease of the dehydrogenase activity and a sudden increase of the protease and lipase activities. The increase of the bacterial stress index also described appropriately the process performance. Regarding the relative abundance of bacterial phylotypes, 37 phyla were identified in the biomass. Proteobacteria were the most abundant (varying the relative abundance between 50.29% and 34.78%) during the first 34 days of operation. From this day on, Bacteroidetes were detected in a greater extent varying the relative abundance of this phylum between 27.20% and 40.45%.

Assessment of azadirachtin-A, a neem tetranortritarpinoid, on rat spermatozoa during in vitro capacitation.

The exploration of the biological assessment of technical azadirachtin, a tetranortritarpinoid from the neem seed kernel, was reviewed. The present study was, therefore, designed to evaluate the dose-dependent in vitro effects of azadirachtin-A, particularly on the functional studies and determination of molecular events, which are critical in the process of sperm capacitation. To assess the effects of the azadirachtin-A on the functional studies, sperm capacitation, the total sperm adenosine triphosphate levels, acrosome reaction (AR), the sperm-egg interaction and the determination of molecular events like cyclic adenosine-3',5'-monophosphate and calcium levels, the appropriate volumes of the sperm suspension were added to the medium to a final concentration of 1×106 sperm/mL and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. The increasing quantities 0.5-2.0 mM/mL and the equivalent volumes of 50% dimethyl sulfoxide were added to the control dishes prior to the addition of spermatozoa and then observed at various time-points for motility and other analyses. Results revealed the dose- and time-dependent decrease in the functional consequence of capacitation, i.e. the percentage of motile spermatozoa, motility score and sperm motility index, levels of molecular events in spermatozoa, followed by declined spontaneous AR leading to lesser binding of the cauda epididymal sperm to the Zona pellucida. The findings confirm the inhibition of rat sperm motility by blocking some biochemical pathways like energy utilization. They also demonstrate that sperm capacitation is associated with the decrease in AR and that the levels of molecular events in spermatozoa can guide us towards the development of a new male contraceptive constituent.

Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

T regulatory cells mediate immunosuppresion by adenosine in peripheral blood, sentinel lymph node and TILs from melanoma patients

T regulatory cells (Tregs), involved in tumour tolerance, can generate Adenosine by CD39/CD73 surface enzymes, which identify four Tregs subsets: CD39+CD73− nTregs, CD39+CD73+ iTregs, CD39−CD73+ oTregs and CD39−CD73− xTregs. In melanoma patients, increased Tregs levels are detected in peripheral blood (PB), sentinel lymph node (SLN) and tumour infiltrating lymphocytes (TILs), but Adenosine role was not investigated yet. We examined total Tregs and Adenosine subsets in PB, SLN and TILs from melanoma patients (n = 32) and PB from healthy donors (HD; n = 10) by flow cytometry. Total Tregs significantly increased in stage III-IV patients PB, in SLN and TILs, as compared to HD/stage I-II patients. Tregs subsets analyses showed that: 1) PB nTregs significantly increased in SLN and decreased in TILs; 2) iTregs significantly increased in stage III-IV patients PB and further significantly increased in SLN and TILs; 3) PB oTregs and xTregs significantly decreased in SLN and TILs. Patients clinical features did not significantly influence total Tregs, except SLN excision order. Results confirmed Tregs role in melanoma progression and indicate Adenosine generation as a novel escape mechanism, being nTregs and iTregs increased in PB/SLN/TILs.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & AimsHuman enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation.MethodsHuman duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s).ResultsDifferentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids.ConclusionsHuman enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

Effects of the antidepressant, mianserin, on early development of fish embryos at low environmentally relevant concentrations

Pharmaceuticals have been considered as emerging organic contaminants in the environment that might pose huge risk to the non-target aquatic organisms. Mianserin, a tetracyclic antidepressant, is present at low detectable concentrations in the aquatic environment; however, limited attention has been devoted to its potential adverse effects on the aquatic animals. In the present study, we first performed an acute toxicity test for mianserin exposure using zebrafish (Danio rerio) embryos during 4–124h post fertilization (hpf). Time-dependent lethal concentrations of mianserin exposure on the zebrafish embryos were firstly determined at mg/L levels. Then, a series of sublethal concentrations of 0.01, 0.1, 1, 10, 100, and 1000μg/L of mianserin were prepared for the short-term exposure of zebrafish embryos for 120h. The results showed that mianserin exposure reduced the body length of zebrafish larvae, in addition to altering multiple physiological and biochemical parameters in the exposed embryos/larvae. A dose-dependent inhibition of the total antioxidant capacity and total cholinesterase activity was revealed in the exposed fish larvae upon increasing the concentrations of mianserin exposure. A U-shaped concentration-dependent response curve was observed for the adrenocorticotropic hormone; however, an inversed U-shaped response curve was obtained for the monoamine oxidase level in response to mianserin exposure. Activities of the total adenosine triphosphatase (T-ATPase), Na+/K+-ATPase, and Ca2+/Mg2+-ATPase were significantly increased in the fish larvae exposed to relatively high doses of mianserin; interestingly however, low dose of mianserin at 10ng/L inhibited their Na+/K+-ATPase and T-ATPase activities. Additionally, the coordinated regulation of cyclic adenosine monophosphate and protein kinase A was observed in the mianserin-exposed fish larvae, implying a reserved signaling pathway involved in the fish response to the antidepressant. Therefore, our study demonstrated that mianserin exposure significantly affected the early development of fish embryos at environmentally relevant concentrations, and suggested that the risk of pharmaceutical contamination of the aquatic environment, even at low doses, should receive more attention.