Use of thioglycolate broth as a pre-analytic transport medium in the diagnosis of prosthetic joint infection.

OBJECTIVES: This study aimed to investigate whether adding tissue samples directly into thioglycolate (TG) broth yielded a greater number of anaerobic organisms than freshly sampled tissue in suspected hip and knee prosthetic joint infections (PJIs). PATIENTS AND METHODS: Between January 2017 and December 2020, a total of 90 patients (46 males, 44 females; median age: 71.7 years; range, 50.8 and 87.8 years) who underwent revision hip or knee arthroplasty were included. Intraoperative samples were taken, with five placed in TG broth and five in standard containers (PC) with subsequent aerobic and anaerobic culturing conducted. Demographic and baseline data of the patients were recorded. The primary outcome was positive bacterial growth from a PJI specimen inoculated directly into TG broth at the time of collection or standard PJI specimen processing. Secondary outcomes investigated were the presence of Cutibacterium acnes (C. acnes) and the curative success of revision procedure. RESULTS: A total of 900 samples (450 PC and 450 TG) were taken from 90 revision arthroplasty patients (47 knees and 43 hips). There was no statistically significant difference in the number of positive bacterial growth samples between TG broth and standard processing (p=0.742). This was consistent with subgroup analysis analyzing C. acnes (p=0.666). CONCLUSION: In hip and knee arthroplasty, there is no benefit in substituting or adding TG broth as a culture medium to better identify both general bacterial species and C. acnes infections specifically. However, the use of TG may be useful in confirming a true positive result for infection.

Tissue-Protective Effect of Erdosteine on Multiple-Organ Injuries Induced by Fine Particulate Matter.

BACKGROUND Fine particulate matter (PM2.5) is the air pollutant that most threatens global public health. The purpose of this study was to observe the inflammatory and oxidative stress injury of multiple organs induced by PM2.5 in rats and to explore the tissue-protective effect of erdosteine. MATERIAL AND METHODS We randomly divided 40 male Wistar rats into a blank control group, a saline group, a PM2.5 exposure group, and an erdosteine intervention group. We assessed changes in organs tissue homogenate and biomarkers of inflammation and oxidative stress in serum and bronchoalveolar lavage fluid (BALF). RESULTS (1) The expressions of IL-6, IL-1ß, TNF-alpha, 8-OHdG, 4-HNE, and PCC in serum and BALF of the PM2.5 exposure group increased, but decreased after treatment with erdosteine, suggesting that erdosteine treatment attenuates inflammatory and oxidative stress injury. (2) The expression of γ-GCS in serum and lungs in the PM2.5 exposure group increased, but did not change significantly after treatment with erdosteine. This suggests that PM2.5 upregulates the level of γ-GCS, while erdosteine does not affect this protective response. (3) The expression of T-AOC in serum, lungs, spleens, and kidneys of the PM2.5 exposure group decreased, but increased after treatment with erdosteine. Our results suggest that PM2.5 can cause imbalance of oxidation/anti-oxidation in multiple organs, and erdosteine can alleviate this imbalance. CONCLUSIONS PM2.5 exposure can lead to inflammatory and oxidative stress damage in serum and organ tissues of rats. Erdosteine may be an effective anti-inflammatory and antioxidant that can reduce this injury.

Effect of Erdosteine on Middle Ear Effusion in Rats by Mediating TLR4 Signaling Pathway.

The study aimed to investigate the effect of erdosteine on middle ear effusion in rats through mediating the Toll-like receptor 4 (TLR4) signaling pathway. Rats were injected with endotoxin to prepare the model of acute secretory otitis media (SOM). Then, they were divided into an acute SOM model group (model group, n = 15) and erdosteine treatment group (18 mg/kg, gavage, treatment group, n = 15). Besides, a normal group (n = 15) was set up. Two weeks later, routine biochemical indicators such as aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were detected. The inflammatory effusion due to otitis media was scored. The content of myeloperoxidase (MPO), matrix metalloproteinase (MMP), and tumor necrosis factor-beta (TNF-ß) in middle ear lavage fluid was detected via enzyme-linked immunosorbent assay (ELISA). Additionally, histomorphological changes were observed with the help of hematoxylin-eosin (HE) staining, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assays were carried out to measure the expression levels of TLR4 pathway genes and proteins as well as the messenger ribonucleic acid (mRNA) expression levels of key factors for otitis media (mucin 2 (MUC2) and MUC5A). In the model group, the levels of AST, ALP, and glutamic-pyruvic transaminase (GPT) were significantly increased (p < 0.05). Besides, the content of MPO, MMP, and TNF-ß was overtly raised in the model group (p < 0.05), while it was notably lowered in the treatment group (p < 0.05). In the treatment group, the cilia were slightly swollen, and inflammatory cells were fewer. The mRNA levels of MUC2, MUC5A, and pathway genes TLR4 and c-Jun N-terminal kinase (JNK) were elevated in the model group. In addition, the protein assay results revealed that the protein levels of TLR4 and JNK were evidently increased in the model group. Erdosteine can treat the middle ear effusion in rats by repressing the activation of the TLR4 signaling pathway.

Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

ATG7 is dispensable for LC3-PE conjugation in thioglycolate-elicited mouse peritoneal macrophages.

Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using atg5-deficient (-/-) and atg7-/-macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited atg7-/- macrophages in response to bafilomycin A1 validated these data. Furthermore, complete loss of ATG3 in atg7-/- macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived atg7-/- macrophages exhibited no LC3-II, even under inflammatory stimuli in vitro. Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data. Abbreviations: ATG: autophagy-related; BM: bone marrow; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PE: phosphatidylethanolamine.

Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer's disease mouse model.

There is increasing evidence that inducing neuronal mitophagy can be used as a therapeutic intervention for Alzheimer's disease. Here, we screen a library of 2024 FDA-approved drugs or drug candidates, revealing UMI-77 as an unexpected mitophagy activator. UMI-77 is an established BH3-mimetic for MCL-1 and was developed to induce apoptosis in cancer cells. We found that at sub-lethal doses, UMI-77 potently induces mitophagy, independent of apoptosis. Our mechanistic studies discovered that MCL-1 is a mitophagy receptor and directly binds to LC3A. Finally, we found that UMI-77 can induce mitophagy in vivo and that it effectively reverses molecular and behavioral phenotypes in the APP/PS1 mouse model of Alzheimer's disease. Our findings shed light on the mechanisms of mitophagy, reveal that MCL-1 is a mitophagy receptor that can be targeted to induce mitophagy, and identify MCL-1 as a drug target for therapeutic intervention in Alzheimer's disease.

Thioredoxin-2 impacts the inflammatory response via suppression of NF-κB and MAPK signaling in sepsis shock.

Sepsis is a progressive disease characterized by excessive inflammatory responses, severe tissue injury and organ dysfunction, ultimately leading to mortality. In this study, we demonstrated that thioredoxin-2 (TRX-2) expression is reduced in macrophages stimulated with lipopolysaccharide (LPS). Overexpression of TRX-2 significantly attenuated interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) production induced by LPS. TRX-2 inhibited LPS-induced inflammatory responses through suppressing activation of the NF-κB and MAPK signaling pathways. Furthermore, TRX-2 induced a significant decrease in mortality in mouse sepsis models in association with reduced inflammatory cytokine production and attenuation of organ injury. Our data collectively support a role of TRX-2 as a critical regulator of sepsis that influences survival by protecting the host from excessive inflammatory damage.

Impact of Lesinurad and allopurinol on experimental Hyperuricemia in mice: biochemical, molecular and Immunohistochemical study.

BACKGROUND: Hyperuricemia is an abnormal increase in uric acid levels in the blood. It is the cause of gout that manifested by inflammatory arthritis and painful disable. Therefore, current study evaluated the potential ameliorative impact of Lesinurad and Allopurinol on the kidneys of hyperuricemic mice at the biochemical, molecular and cellular levels. METHODS: Lesinurad and allopurinol alone or in combination were orally administered to hyperuricemic and control mice for seven consecutive days. Levels of uric acid and blood urea nitrogen, along with antioxidants and inflammatory cytokines (IL-1ß and TNF-α) were measured in the serum. The mRNA expression of mouse urate anion transporter-1, glucose transporter 9, organic anion transporters, in renal tissues were examined using quantitative real time PCR. Simultaneously, the immunoreactivity of transforming growth factor-beta 1 was examined immunohistochemically. RESULTS: Lesinurad and allopurinol administration resulted in significant decrease in serum levels of uric acid, blood urea nitrogen, xanthine oxidase activity, catalase, glutathione peroxidase and inflammatory cytokines (IL-1ß and TNF-α) reported in hyperuricemic mice. Both partially reversed oxonate-induced alterations in renal mURAT-1, mGLUT-9, mOAT-1 and mOAT-3 expressions, as well as alterations in the immunoreactivity of TGF- ß1, resulting in the increase of renal uric acid secretion and excretion. The combined administration of lesinurad and ALP restored all altered parameters in a synergistic manner, improving renal function in the hyperuricemic mouse model employed. CONCLUSION: This study confirmed synergistic ameliorative hypouricemic impact of both lesinurad and allopurinol in the treatment of hyperuricemia in mice at the biochemical, molecular and cellular levels.

Dinuclear cobalt and nickel complexes of a mercaptoacetic acid substituted 1,2,4-triazole ligand: syntheses, structures and urease inhibitory studies.

Because of its versatile coordination modes and strong coordination ability, the mercaptoacetic acid substituted 1,2,4-triazole 2-{[5-(pyridin-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetic acid (H2L) was synthesized and characterized. Treatment of H2L with cobalt and nickel acetate afforded the dinuclear complexes {µ-3-[(carboxylatomethyl)sulfanyl]-5-(pyridin-2-yl)-4H-1,2,4-triazol-4-ido-κ2N1,N5:N2,O}bis[aqua(methanol-κO)cobalt(II)] methanol disolvate, [Co2(C9H6N4O2S)2(CH3OH)2(H2O)2]·2CH3OH (1), and {µ-3-[(carboxylatomethyl)sulfanyl]-5-(pyridin-2-yl)-4H-1,2,4-triazol-4-ido-κ2N1,N5:N2,O}bis[diaquanickel(II)] methanol disolvate dihydrate, [Ni2(C9H6N4O2S)2(H2O)4]·2CH3OH·2H2O (2), respectively. Complex 1 crystallized in the monoclinic space group P21/c, while 2 crystallized in the tetragonal space group I41/a. Single-crystal X-ray diffraction studies revealed that H2L is doubly deprotonated and acts as a tetradentate bridging ligand in complexes 1 and 2. For both of the obtained complexes, extensive hydrogen-bond interactions contribute to the formation of their three-dimensional supermolecular structures. Hirshfeld surface analysis was used to illustrate the intermolecular interactions. Additionally, the urease inhibitory activities of 1, 2 and H2L were investigated against jack bean urease, where the two complexes revealed strong urease inhibition activities.

Thiol-Based Drugs in Pulmonary Medicine: Much More than Mucolytics.

Thiol-based drugs are considered as mucolytics because they decrease the viscosity and mostly decrease the elasticity of bronchial secretions by reducing disulfide bonds in proteins. However, they can also act as antioxidant drugs directly through free sulfhydryl groups that serve as a source of reducing equivalents, as well as indirectly through the replenishment of intracellular glutathione (GSH) levels. Modulation of neurokinin A levels may also be related to the effect of thiol drugs on oxidative stress. Moreover, thiol-based drugs interfere with inflammatory pathways and modulate human bronchial tone. They might also be considered as therapeutic agents against some types of infection because they reduce bacterial adhesion to the respiratory epithelial cell surface and inhibit biofilm formation, causing biofilm disruption and thereby improving the efficacy of antibiotic therapy.

Confounding off-target effects of BH3 mimetics at commonly used concentrations: MIM1, UMI-77, and A-1210477.

Targeting anti-apoptotic BCL2 family proteins has become an attractive therapeutic strategy for many cancers, and the BCL2-selective inhibitor ABT-199 (venetoclax) has obtained clinical success. However, MCL1 can promote drug resistance and overall cancer cell survival. Thus, there is a critical need to develop an effective drug that antagonizes MCL1. However, most putative MCL1 inhibitors have been misclassified as they fail to directly inhibit MCL1 in cells, but rather induce the pro-apoptotic protein NOXA. We have investigated three putative MCL1 inhibitors: MIM1, UMI-77, and A-1210477. All three compounds were developed in cell-free assays and then found to be cytotoxic, and hence assumed to directly target MCL1 in cells. Here, we investigated whether these compounds directly inhibit MCL1 or inhibit MCL1 indirectly through the induction of NOXA. Both MIM1- and UMI-77-induced NOXA through the unfolded protein response pathway, and sensitized leukemia cells to ABT-199; this cytotoxicity was dependent on NOXA suggesting that these compounds do not directly target MCL1. A-1210477 was the only compound that did not induce NOXA, but it still sensitized cells to ABT-199. A-1210477 induced accumulation of MCL1 protein consistent with it binding and preventing MCL1 degradation. However, at concentrations used in several prior studies, A-1210477 also induced cytochrome c release, caspase activation, and apoptosis in a BAX/BAK-independent manner. Furthermore, the release of cytochrome c occurred without loss of mitochondrial membrane potential. This apoptosis was extremely rapid, sometimes occurring within 0.5-1 h. Hence, we have identified a novel mechanism of apoptosis that circumvents the known mechanisms of cytochrome c release. It remains to be determined whether these unexpected mechanisms of action of putative BH3 mimetics will have therapeutic potential.

Characterization of Stereoselective Metabolism, Inhibitory Effect on Uric Acid Uptake Transporters, and Pharmacokinetics of Lesinurad Atropisomers.

Lesinurad [Zurampic; 2-(5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-ylthio)], a selective inhibitor of uric acid reabsorption transporters approved for the treatment of gout, is a racemate of two atropisomers. The objective of this investigation was to evaluate the stereoselectivity of metabolism, the inhibitory potency on kidney uric acid reabsorption transporters (URAT1 and OAT4), and the clinical pharmacokinetics of the lesinurad atropisomers. Incubations with human liver microsomes (HLM), recombinant CYP2C9, and recombinant CYP3A4 were carried out to characterize the stereoselective formation of three metabolites: M3 (hydroxylation), M4 (a dihydrodiol metabolite), and M6 (S-dealkylation). The formation of M3 in HLM with atropisomer 1 was approximately twice as much as that with atropisomer 2, whereas formation of M4 with atropisomer 1 was 8- to 12-fold greater than that with atropisomer 2. There were no significant differences in the plasma protein binding among lesinurad and the atropisomers. Following oral administration of 400 mg lesinurad once daily for 14 days to healthy human volunteers, the systemic exposure (C max at steady state and area under the concentration-time curve from time zero to the time of dosing interval) of atropisomer 1 was approximately 30% lower than that of atropisomer 2, whereas renal clearance was similar. In vitro cell-based assays using HEK293 stable cells expressing URAT1 and OAT4 demonstrated that atropisomer 2 was approximately 4-fold more potent against URAT1 than atropisomer 1 and equally active against OAT4. In conclusion, lesinurad atropisomers showed stereoselectivity in clinical pharmacokinetics, metabolism, and inhibitory potency against URAT1.

Investigation of Therapeutic Effects of Erdosteine on Polycystic Ovary Syndrome in a Rat Model.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a serious endocrine disorder. In the present study, we investigated the therapeutic effects of erdosteine in letrozole-induced PCOS in rats. METHODS: Thirty-two Wistar albino female rats were grouped as control group (C), PCOS group (PCOS), PCOS-metformin group (PCOS+MET), and PCOS-erdosteine group (PCOS+Erd). PCOS was induced by administering letrozole; such rats presented with sex hormone disorder, abnormal estrous cycles determined by daily vaginal smear, large cystic follicles, and increasing fasting insulin levels. After induction of PCOS, metformin (500 mg/kg/day) and erdosteine (100 mg/kg/day) were given orally to the treatment groups for 30 days. Serum concentrations of glucose, total cholesterol, low- and high-density lipoprotein, triglyceride, as well as the total oxidant and antioxidant status, oxidative stress index, circulating estrone (E1), estradiol (E2), testosterone, and androstenedione were evaluated. The ovaries were graded histologically. RESULTS: Weights of ovarian tissues (p < 0.05) and the number of atretic follicles (p < 0.001) and cystic follicles (p < 0.01) decreased in the PCOS+Erd group; the corpus luteum number was significantly higher in the PCOS+Erd group (p < 0.01) as compared with the PCOS group. Lipid parameters (total-C, LDL-C, and TG), E1 (estrone), E1/E2 ratio, testosterone, and androstenedione significantly decreased, while HDL-C and E2 (estradiol) significantly increased in the PCOS+Erd group as compared with the PCOS group. Moreover glucose, insulin, and HOMA-IR were reduced with treatment of erdosteine (p > 0.05, p < 0.001, and p < 0.001, respectively). CONCLUSION: It is suggested that erdosteine may be used in the treatment of PCOS as an alternative to metformin. It appears that our findings might be supported by clinical and molecular studies.

Urate transporter inhibitor lesinurad is a selective peroxisome proliferator-activated receptor gamma modulator (sPPARγM) in vitro.

Gout is the most common arthritic disease in human but was long neglected and therapeutic options are not satisfying. However, with the recent approval of the urate transporter inhibitor lesinurad, gout treatment has experienced a major innovation. Here we show that lesinurad possesses considerable modulatory potency on peroxisome proliferator-activated receptor γ (PPARγ). Since gout has a strong association with metabolic diseases such as type 2 diabetes, this side-activity appears as very valuable contributing factor to the clinical efficacy profile of lesinurad. Importantly, despite robustly activating PPARγ in vitro, lesinurad lacked adipogenic activity, which seems due to differential coactivator recruitment and is characterized as selective PPARγ modulator (sPPARγM).

Allosteric and Orthosteric Activators of mGluR8 Differentially Affect the Chemotherapeutic-Induced Human Neuroblastoma SH-SY5Y Cell Damage: The Impact of Cell Differentiation State.

The participation of group III metabotropic glutamate receptors (mGluRs) in cancer growth and progression is still an understudied issue. Based on our recent data on high expression of mGluR8 in human neuroblastoma SH-SY5Y cells, in this study, we evaluated the effect of an mGluR8-specific positive allosteric modulator (PAM: AZ12216052) and orthosteric agonist [(S)-3,4-DCPG ((S)-3,4-dicarboxyphenylglycine)] on chemotherapeutic (doxorubicin, irinotecan or cisplatin)-evoked cell damage in undifferentiated (UN-) and retinoic acid-differentiated (RA-) SH-SY5Y cells. The data showed that AZ12216052 as well as a group III mGluR antagonist (UBP1112) but not (S)-3,4-DCPG partially inhibited the cell damage evoked by doxorubicin, irinotecan or cisplatin in UN-SH-SY5Y cells. In RA-SH-SY5Y, we observed only a modest protective effect of mGluR8 PAM. In contrast, both types of mGluR8 activators significantly enhanced toxic effects of doxorubicin and irinotecan in RA-SH-SY5Y cells. These data suggest that in undifferentiated neuroblastoma malignant cells, some mGluR8 modulators can decrease cytotoxic effects of chemotherapeutics which exclude them from the group of putative anticancer agents. On the other hand, in SH-SY5Y cells differentiated to a more mature neuron-like phenotype, that is non-malignant cells, the mGluR8 activators can aggravate the chemotherapeutic neurotoxicity which is a well-known undesired effect of these drugs. Our pharmacological data add new observations to the unexplored field of research on the role of mGluR8 in cancer, pointing to complexity of response which could be mediated by particular types of mGluR8 ligands at least in neuroblastoma cells.

Systematic Structure-Activity Relationship (SAR) Exploration of Diarylmethane Backbone and Discovery of A Highly Potent Novel Uric Acid Transporter 1 (URAT1) Inhibitor.

In order to systematically explore and better understand the structure-activity relationship (SAR) of a diarylmethane backbone in the design of potent uric acid transporter 1 (URAT1) inhibitors, 33 compounds (1a-1x and 1ha-1hi) were designed and synthesized, and their in vitro URAT1 inhibitory activities (IC50) were determined. The three-round systematic SAR exploration led to the discovery of a highly potent novel URAT1 inhibitor, 1h, which was 200- and 8-fold more potent than parent lesinurad and benzbromarone, respectively (IC50 = 0.035 µM against human URAT1 for 1h vs. 7.18 µM and 0.28 µM for lesinurad and benzbromarone, respectively). Compound 1h is the most potent URAT1 inhibitor discovered in our laboratories so far and also comparable to the most potent ones currently under development in clinical trials. The present study demonstrates that the diarylmethane backbone represents a very promising molecular scaffold for the design of potent URAT1 inhibitors.

Effects of Erdosteine on Experimental Acute Pancreatitis Model.

OBJECTIVE: To create acute pancreatitis condition experimentally in rats using cerulein, and to reveal histopathological effects in pancreatic tissue with erdosteine. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of General Surgery, Duzce University, Turkey, from June to October 2014. METHODOLOGY: Thirty male Wistar albino rats were divided into three groups. No procedures were applied to Group 1. The rats in Group 2 and Group 3 were injected cerulein, to establish an experimental pancreatitis model and the blood amylase and lipase values were examined. The rats in Group 3 were given 10 mg/kg erdosteine. This treatment was continued for another 2 days and the rats were sacrificed. The pancreatic tissues were examined histopathologically for edema, inflammation, acinar necrosis, fat necrosis, and vacuolization. RESULTS: The lipase and amylase values and the histopathological examination of pancreatic tissues evidenced that the experimental acute pancreatitis model was established and edema, inflammation, acinar necrosis, fat necrosis, and vacuolization were observed in the pancreatic tissues. The statistical results suggest that erdosteine can decrease the edema, inflammation, acinar necrosis, fat necrosis and vacuolization scores in the tissues. CONCLUSION: The severity of acute pancreatitis, induced by cerulein in rats, is reduced with the use of erdosteine.

Identification and characterization of a potent and selective inhibitor of human urate transporter 1.

BACKGROUND: Selective inhibitors of human urate transporter 1 (hURAT1) are considered to be effective treatment for hyperuricemia and gout, which can reduce the reabsorption of more than 90% of uric acid in the proximal tubule of the kidney. We aimed to design and synthesize a more potent hURAT1 based on the structure of Lesinurad (LU), which was reported to lower uric acid levels with IC50 value of hURAT1 (about 60µM). METHODS: A cell model was conducted and characterized via Real-time qRCR and Western blot. We synthesized and identified a new midazole analogue of LU. Cells stably-expressing hURAT1 or human organic anion transporter 1 (hOAT1) were used in the [14C] urate or 6-carboxyfluorescein (6-CF) uptake assays to test the activities of the newly synthesized compound. The uric acid lowering effects of LU and LUM and their effects on urea nitrogen and creatinine in potassium oxonate-induced hyperuricemic rats were analyzed. RESULTS: The [14C] Urate uptake assay using hURAT1 stably transfected MDCK cells indicated that LUM was more potent than LU against hURAT1, with IC50 values of 3.22µM and 65.47µM, respectively. LU and LUM also effectively suppressed hOAT1-mediated 6-CF uptake, and the IC50 hURAT1/IC50 hOAT1 of LU and LUM was1.49 and 0.35 respectively, indicating a better selectivity for LUM than LU. In vivo, LUM-Na (40mg/kg) showed more potent activity in reducing serum uric acid levels in potassium oxonate-induced hyperuricemic rats, compared to similar doses of LU-Na. CONCLUSION: LUM was demonstrated to be as potent a uricosuric drug as LU.

Intracellular Chloride Regulation in AVP+ and VIP+ Neurons of the Suprachiasmatic Nucleus.

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl-]i). Here, using ratiometric Cl- imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl-]i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl-]i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl-]i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl-]i in SCN neurons. Interestingly, we observed that [Cl-]i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.

Activation of spinal alpha-7 nicotinic acetylcholine receptor shortens the duration of remifentanil-induced postoperative hyperalgesia by upregulating KCC2 in the spinal dorsal horn in rats.

Background Accumulating evidence has shown that the signal from spinal brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 plays a critical role in the process of pain hypersensitivity. The activation of alpha-7 nicotinic acetylcholine receptors could have an analgesic effect on remifentanil-induced postoperative hyperalgesia. Nevertheless, whether intrathecal administration of PNU-120596, an alpha-7 nicotinic acetylcholine receptors selective type II positive allosteric modulator, before surgery could affect the duration of remifentanil-induced postoperative hyperalgesia remains unknown, and the effects of alpha-7 nicotinic acetylcholine receptors activation on the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal in the spinal dorsal horn of rats with remifentanil-induced postoperative hyperalgesia is still enigmatic. Results We demonstrated that the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal played a critical role in the development of remifentanil-induced postoperative hyperalgesia. Intrathecal administration of PNU-120596 (8 µg/kg, 15 min before surgery) was associated with earlier signs of recovery from remifentanil-induced postoperative hyperalgesia. Simultaneously, remifentanil-induced postoperative hyperalgesia-induced K+-Cl- cotransporter-2 downregulation was partly reversed and coincided with a decreased expression of brain-derived neurotrophic factor/tyrosine receptor kinase B in the spinal dorsal horn, approximately correlating with the time course of the nociceptive behavior. Moreover, intrathecal administration of the K+-Cl- cotransporter-2 inhibitor VU0240551 significantly reduced the analgesic effect of PNU-120596 on remifentanil-induced postoperative hyperalgesia. Conclusions The activation of alpha-7 nicotinic acetylcholine receptors induced a shorter duration of remifentanil-induced postoperative hyperalgesia by restoring the brain-derived neurotrophic factor/tyrosine receptor kinase B-K+-Cl- cotransporter-2 signal in the spinal dorsal horn of rats, which provides new insight into treatment in clinical postoperative pain management.