Current Drug Development Overview: Targeting Voltage-Gated Calcium Channels for the Treatment of Pain.

Voltage-gated calcium channels (VGCCs) are targeted to treat pain conditions. Since the discovery of their relation to pain processing control, they are investigated to find new strategies for better pain control. This review provides an overview of naturally based and synthetic VGCC blockers, highlighting new evidence on the development of drugs focusing on the VGCC subtypes as well as mixed targets with pre-clinical and clinical analgesic effects.

Serum levels of inflammatory markers and HbA1c in patients with type 2 diabetes and apical periodontitis: Preliminary findings.

This pilot study assessed the glycaemic control and the serum levels of inflammatory mediators in type 2 diabetes (T2DM) patients with apical periodontitis (AP). Thirty individuals were divided into four groups: Healthy (H); with AP (AP); with T2DM (T2DM); and with T2DM and AP (T2DM-AP). Demographic and pharmacological data were registered. The body mass index (BMI) and the levels of glycated haemoglobin (HbA1c) and IL-1ß, IL-6, IL-10, CCL3 and CCL4 were evaluated. AP areas were determined radiographically. Mean age was 64 ± 12 years, with 63% females. Most T2DM patients were under treatment with metformin and antihypertensives. BMI and H1bAc were significantly higher in T2DM patients in relation to H and AP groups. The AP areas were larger in the T2DM-AP group, compared with the AP group. These preliminary findings suggest no influence of AP on glycaemic control or inflammatory levels amongst T2DM patients, although T2DM increased the AP severity.

Blockade of the kinin B1 receptor counteracts the depressive-like behaviour and mechanical allodynia in ovariectomised mice.

Menopause is related to a decline in ovarian oestrogen production, affecting the perception of the somatosensory stimuli, changing the immune-inflammatory systems, and triggering depressive symptoms. It has been demonstrated that the inhibition of the kinin B1 and B2 receptors (B1R and B2R) prevented the depressive-like behaviour and the mechanical allodynia that was induced by immune-inflammatory mediators in mice. However, there is no evidence regarding the role of the kinin receptors in the depressive-like and nociceptive behaviour in female mice that were subjected to bilateral ovariectomy (OVX). This study has shown that the OVX mice developed time-related mechanical allodynia, together with an increased immobility time as indicative of depression. Both of these changes were reduced by the genetic deletion of B1R, or by the pharmacological blockade of the selective kinin B1R antagonist R-715 (acute, i.p.). The genetic deletion or the pharmacological inhibition of B2R (HOE 140, i.p.) did not prevent the OVX-elicited behavioural changes. The data has suggested a particular modulation of kinin B1R in the nociceptive and depressive-like behaviour in the OVX mice. The selective inhibition of the B1R receptor may be a new pharmacological target for treating pain and depression symptoms in women during the perimenopause/menopause period.

High-fat diet effect on periapical lesions and hepatic enzymatic antioxidant in rats.

AIMS: To evaluate the effects of a high-fat diet (HFD) on the progression of apical periodontitis (AP), local inflammation, systemic antioxidant status, and blood lipid profile in rats. MAIN METHODS: Sixteen male Wistar rats were fed a standard diet (SD) or a HFD. At the sixth experimental week, the pulp chambers of the mandibular first molars were exposed to develop AP. A glucose tolerance test was performed the week before euthanasia. At the tenth experimental week, the animals were euthanized and the livers were collected to estimate catalase (CAT) and reduced glutathione (GSH) levels. Blood was acquired for biochemical analysis. The size of AP was estimated from radiographs and described as AP size-to-body weight ratio; inflammatory grade of AP was determined by histological analysis. KEY FINDINGS: At the end of the experimental period, the rats fed the HFD had 30% less weight (P < 0.0001) and higher blood glucose levels after 30 min of sucrose intake (P < 0.05) than those fed the SD. Animals from the HFD group had lower levels of CAT (P < 0.01), but the same was not observed in the GSH levels. Plasma insulin and total cholesterol were not affected by the diet. The rats fed the HFD presented greater AP than those fed the SD (P < 0.05). However, the local inflammatory infiltrate was similar in both groups. SIGNIFICANCE: The alterations promoted by the consumption of a HFD were not only observed systemically, but also locally, producing greater AP in rats than a SD.

Phoneutria nigriventer Tx3-3 peptide toxin reduces fibromyalgia symptoms in mice.

Fibromyalgia is characterized by the amplification of central nervous system pain with concomitant fatigue, sleep, mood disorders, depression, and anxiety. It needs extensive pharmacological therapy. In the present study, Swiss mice were treated with reserpine (0.25 mg/kg, s.c.) over three consecutive days, in order to reproduce the pathogenic process of fibromyalgia. On day 4, the administrations of the Tx3-3 toxin produced significant antinociception in the mechanical allodynia (87.16% ±12.7%) and thermal hyperalgesia (49.46% ± 10.6%) tests when compared with the PBS group. The effects produced by the classical analgesics (duloxetine 30 mg/kg, pramipexole 1 mg/kg, and pregabalin 30 mg/kg, p.o., respectively) in both of the tests also demonstrated antinociception. The administrations were able to increase the levels of the biogenic amines (5-HTP and DE) in the brain. The treatments with pramipexole and pregabalin, but not duloxetine, decreased the immobility time in the FM-induced animals that were submitted to the forced swimming test; however, the Tx3-3 toxin (87.45% ± 4.3%) showed better results. Taken together, the data has provided novel evidence of the ability of the Tx3-3 toxin to reduce painful and depressive symptoms, indicating that it may have significant potential in the treatment of FM.

Salivary Gland Tumors: a Ten-Year Retrospective Analysis in a Brazilian Teaching Hospital / Tumores de las Glándulas Salivales: Análisis Retrospectivo de Diez Años en un Hospital de Enseñanza Brasileño / Tumores de Glândulas Salivares: Análise Retrospectiva de Dez Anos em um Hospital Escola Brasileiro

Introduction: The tumors affecting salivary glands have a wide morphological diversity. Objective: This study aimed to examine the prevalence of salivary gland tumors in patients treated at São Lucas Teaching Hospital at the Pontifical Catholic University of Rio Grande do Sul (HSL-PUCRS), in Porto Alegre (RS), Brazil, from 2007 to 2016. Method: A retrospective study analyzing 201 files from the Department of Pathology at the HSL-PUCRS was carried out, by revising the medical records. Results: Seventy-three cases of salivary gland tumors were found, and their electronic and physical medical records were analyzed. Of the 73 cases, 56 (76.7%) were benign tumors and 17 (23.3%) were malignant tumors. The age group with the highest number of cases was between 41 and 60 years of age and the highest prevalence was found in females, with 54.8% of the cases. The parotid gland presented the highest prevalence, accounting for 72.6% of the cases. The predominant neoplasia was the pleomorphic adenoma, accounting for 53.4% of the tumors. The standard of distribution of neoplasms of salivary glands was similar to the encountered in other Brazilian regions. Conclusion: The largest salivary glands were the most affected by neoplastic processes. Pleomorphic adenoma and adenoid cystic carcinoma were the most frequent benign and malignant tumors, respectively, and parotid gland was the most affected site. In the light of previous literature data, the results allow to infer that some demographic characteristics (for example, sex and age) vary among the different geographic regions

Introducción: Los tumores que afectan a las glándulas salivales tienen una amplia diversidad morfológica. Objetivo: Identificar la prevalencia de neoplasias de glándulas salivales en pacientes atendidos en el Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul (HSLPUCRS), en Porto Alegre (RS), desde 2007 hasta 2016. Método: Estudio retrospectivo mediante el análisis de 201 registros del Departamento de Patología de HSL-PUCRS. Resultados: Se encontraron 73 casos de neoplasias de glándulas salivales y se analizaron los registros electrónicos y físicos de los casos seleccionados. De los 73 casos, 56 (76,7%) fueron de neoplasias benignas y 17 (23,3%) de neoplasias malignas. El grupo de edad con mayor número de casos fue el de 41 a 60 años, y la mayor prevalencia en mujeres, con 54,8%. La glándula parótida tuvo una mayor prevalencia, constituyendo 72,6% de los casos. El tipo neoplásico más prevalente fue el adenoma pleomorfo, con 53,4%. El patrón de distribución de las neoplasias de glándulas salivales fue similar al encontrado en otras regiones de Brasil. Conclusión: Las glándulas salivales mayores fueron las glándulas más afectadas por procesos neoplásicos. El adenoma pleomórfico y el carcinoma adenoide quístico fueron los tumores benignos y malignos más frecuentes, respectivamente y el sitio más afectado fue la glándula parótida. Con base en la literatura previa, estos resultados permiten inferir que algunas características demográficas (por ejemplo, sexo y edad) varían entre las distintas regiones geográficas

Introdução: Os tumores que afetam as glândulas salivares apresentam vasta diversidade morfológica. Objetivo: Identificar a prevalência de neoplasias de glândulas salivares em pacientes atendidos no Hospital São Lucas da Pontifícia Universidade Católica do Rio Grande do Sul (HSL-PUCRS), em Porto Alegre (RS), no período de 2007 a 2016. Método: Estudo retrospectivo por meio da análise de 201 arquivos do Departamento de Patologia do HSL-PUCRS. Resultados: Foram encontrados 73 casos de neoplasias de glândulas salivares e os prontuários eletrônicos e físicos dos casos selecionados foram analisados. Dos 73 casos, 56 (76,7%) eram de neoplasias benignas e 17 (23,3%) de neoplasias malignas. A faixa etária com maior número de casos foi entre 41 e 60 anos e o sexo feminino apresentou a maior prevalência com 54,8%. A glândula parótida apresentou maior prevalência, perfazendo 72,6% dos casos. O tipo neoplásico mais prevalente foi o adenoma pleomórfico, com 53,4%. O padrão de distribuição das neoplasias de glândulas salivares foi semelhante ao encontrado em outras Regiões do Brasil. Conclusão: As glândulas salivares maiores foram as mais afetadas pelos processos neoplásicos. Adenoma pleomórfico e carcinoma adenoide cístico foram os tumores benignos e malignos mais frequentes, respectivamente, e a glândula parótida foi o local mais acometido. Com base na literatura prévia, esses resultados permitem inferir que algumas características demográficas (por exemplo, sexo e idade) variam entre as diferentes Regiões geográficas

Neural Regenerative Potential of Stem Cells Derived from the Tooth Apical Papilla.

The regenerative effects of stem cells derived from dental tissues have been previously investigated. This study assessed the potential of human tooth stem cells from apical papilla (SCAP) on nerve regeneration. The SCAP collected from nine individuals were characterized and polarized by exposure to interferon-γ (IFN-γ). IFN-γ increased kynurenine and interleukin-6 (IL-6) production by SCAP, without affecting the cell viability. IFN-γ-primed SCAP exhibited a decrease of brain-derived neurotrophic factor (BDNF) mRNA levels, followed by an upregulation of glial cell-derived neurotrophic factor mRNA. Ex vivo, the co-culture of SCAP with neurons isolated from the rat dorsal root ganglion induced neurite outgrowth, accompanied by increased BDNF secretion, irrespective of IFN-γ priming. In vivo, the local application of SCAP reduced the mechanical and thermal hypersensitivity in Wistar rats that had been submitted to sciatic chronic constriction injury. The SCAP also reduced the pain scores, according to the evaluation of the Grimace scale, partially restoring the myelin damage and BDNF immunopositivity secondary to nerve lesion. Altogether, our results provide novel evidence about the regenerative effects of human SCAP, indicating their potential to handle nerve injury-related complications.

Taxane-induced neurotoxicity: Pathophysiology and therapeutic perspectives.

Taxane-derived drugs are antineoplastic agents used for the treatment of highly common malignancies. Paclitaxel and docetaxel are the most commonly used taxanes; however, other drugs and formulations have been used, such as cabazitaxel and nab-paclitaxel. Taxane treatment is associated with neurotoxicity, a well-known and relevant side effect, very prevalent amongst patients undergoing chemotherapy. Painful peripheral neuropathy is the most dose-limiting side effect of taxanes, affecting up to 97% of paclitaxel-treated patients. Central neurotoxicity is an emerging side effect of taxanes and it is characterized by cognitive impairment and encephalopathy. Besides impairing compliance to chemotherapy treatment, taxane-induced neurotoxicity (TIN) can adversely affect the patient's life quality on a long-term basis. Despite the clinical relevance, not many reviews have comprehensively addressed taxane-induced neurotoxicity when they are used therapeutically. This article provides an up-to-date review on the pathophysiology of TIN and the novel potential therapies to prevent or treat this side effect.

Nociceptin/orphanin FQ receptor modulates painful and fatigue symptoms in a mouse model of fibromyalgia.

Generalized pain and fatigue are both hallmarks of fibromyalgia, a syndrome with an indefinite etiology. The treatment options for fibromyalgia are currently limited, probably because of its intricate pathophysiology. Thus, further basic and clinical research on this condition is currently needed. This study investigated the effects of nociceptin/orphanin FQ (N/OFQ) receptor (NOPr) ligands and the modulation of the NOP system in the preclinical mouse model of reserpine-induced fibromyalgia. The effects of administration of the natural agonist N/OFQ and the selective NOPr antagonists (UFP-101 and SB-612111) were evaluated in fibromyalgia-related symptoms in reserpine-treated mice. The expression of prepronociceptin/orphanin FQ and NOPr was assessed in central and peripheral sites at different time points after reserpine administration. Nociceptin/orphanin FQ displayed dual effects in the behavioral changes in the reserpine-elicited fibromyalgia model. The peptide NOPr antagonist UFP-101 produced analgesic and antifatigue effects, by preventing alterations in brain activity and skeletal muscle metabolism, secondary to fibromyalgia induction. The nonpeptide NOPr antagonist SB-612111 mirrored the favorable effects of UFP-101 in painful and fatigue alterations induced by reserpine. A time-related up- or downregulation of prepronociceptin/orphanin FQ and NOPr was observed in supraspinal, spinal, and peripheral sites of reserpine-treated mice. Our data shed new lights on the mechanisms underlying the fibromyalgia pathogenesis, supporting a role for N/OFQ-NOP receptor system in this syndrome.

Design of Novel Inhibitors of Human Thymidine Phosphorylase: Synthesis, Enzyme Inhibition, in Vitro Toxicity, and Impact on Human Glioblastoma Cancer.

Overexpressed human thymidine phosphorylase (hTP) has been associated with cancer aggressiveness and poor prognosis by triggering proangiogenic and antiapoptotic signaling. Designed as transition-state analogues by mimicking the oxacarbenium ion, novel pyrimidine-2,4-diones were synthesized and evaluated as inhibitors of hTP activity. The most potent compound (8g) inhibited hTP in the submicromolar range with a noncompetitive inhibition mode with both thymidine and inorganic phosphate substrates. Furthermore, compound 8g was devoid of apparent toxicity to a panel of mammalian cells, showed no genotoxicity signals, and had low probability of drug-drug interactions and moderate in vitro metabolic rates. Finally, treatment with 8g (50 mg/(kg day)) for 2 weeks (5 days/week) significantly reduced tumor growth using an in vivo glioblastoma model. To the best of our knowledge, this active compound is the most potent in vitro hTP inhibitor with a kinetic profile that cannot be reversed by the accumulation of any enzyme substrates.

Stability and efficacy of combined nystatin and chlorhexidine against suspensions and biofilms of Candida albicans.

OBJECTIVE: Nystatin and chlorhexidine are extensively used in oral medicine; however, there is some controversy about the possibility of these drugs showing antagonism. To clarify this issue, this study investigated the efficacy and stability of nystatin and chlorhexidine in combination. DESIGN: An in vitro study was conducted to analyze the effect of nystatin and chlorhexidine combined on Candida albicans ATCC 18804, using the drugs mixed as a single formulation and as independent formulations used sequentially with different time intervals between them. The minimum inhibitory concentration (MIC) and effects on C. albicans suspensions and biofilms were evaluated. Also, the stability of nystatin and chlorhexidine in a mixture was tested by high performance liquid chromatography (HPLC). RESULTS: When nystatin and chlorhexidine were mixed in a single formulation, there was no significant difference in MIC compared to that of the drugs used alone (as the only treatment). However, when these drugs were used as independent formulations, sequentially with time intervals in between, their MICs were higher than the respective MIC of the drug used alone, except for the MIC of chlorhexidine with a 10-min interval. Nystatin/chlorhexidine combinations showed lower activity against C. albicans biofilms, except for that with a 30-min interval. The drugs when combined showed high percentages of degradation at all the times evaluated. CONCLUSIONS: The combination of nystatin and chlorhexidine seems to interfere with the efficacy of the drugs and to increase their rate of degradation.

Design and optimization of biocompatible polycaprolactone/poly (l-lactic-co-glycolic acid) scaffolds with and without microgrooves for tissue engineering applications.

This study investigated the effects of smooth and microgrooved membrane blends, with different polycaprolactone (PCL)/ poly(lactic-co-glycolic acid) (PLGA) ratios on the viability, proliferation, and adhesion of different mammalian cell types. The polymer matrices with and without microgrooves, obtained by solvent casting, were characterized by field-emission scanning electron microscopy, atomic force microscopy, contact angle and Young's modulus. Blend characterization showed an increase in roughness and stiffness of membranes with 30% PLGA, without any effect on the contact angle value. Pure PCL significantly decreased the viability of Vero, HaCaT, RAW 264.7, and human fetal lung and gingival fibroblast cells, whereas addition of increasing concentrations of PLGA led to a reduced cytotoxicity. Increased proliferation rates were observed for all cell lines. Fibroblasts adhered efficiently to smooth membranes of the PCL70/PLGA30 blend and pure PLGA, compared to pure PCL and silicone. Microgrooved membranes promoted similar cell adhesion for all groups. Microstructured membranes (15 and 20-µm wide grooves) promoted suitable orientation of fibroblasts in both PCL70/PLGA30 and pure PLGA, as compared to pure PCL. Neuronal cells of the dorsal root ganglion exhibited an oriented adhesion to all the tested microgrooved membranes. Data suggest a satisfactory safety profile for the microgrooved PCL70/PLGA30 blend, pointing out this polymer combination as a promising biomaterial for peripheral nerve regeneration when cell orientation is required. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1522-1534, 2018.

Pre-clinical effects of metformin and aspirin on the cell lines of different breast cancer subtypes.

Background Breast cancer is highly prevalent among women worldwide. It is classified into three main subtypes: estrogen receptor positive (ER+), human epidermal growth factor receptor 2 positive (HER2+), and triple negative breast cancer (TNBC). This study has evaluated the effects of aspirin and metformin, isolated or in a combination, in breast cancer cells of the different subtypes. Methods The breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 were treated with aspirin and/or metformin (0.01 mM - 10 mM); functional in vitro assays were performed. The interactions with the estrogen receptors (ER) were evaluated in silico. Results Metformin (2.5, 5 and 10 mM) altered the morphology and reduced the viability and migration of the ER+ cell line MCF-7, whereas aspirin triggered this effect only at 10 mM. A synergistic effect for the combination of metformin and aspirin (2.5, 5 or 10 mM each) was observed in the TNBC cell subtype MDA-MB-231, according to the evaluation of its viability and colony formation. Partial inhibitory effects were observed for either of the drugs in the HER2+ cell subtype SK-BR-3. The effects of metformin and aspirin partly relied on cyclooxygenase-2 (COX-2) upregulation, without the production of lipoxins. In silico, metformin and aspirin bound to the ERα receptor with the same energy. Conclusion We have provided novel evidence on the mechanisms of action of aspirin and metformin in breast cancer cells, showing favorable outcomes for these drugs in the ER+ and TNBC subtypes.

Characterization of a rat model with temporomandibular joint osteoarthritis following a surgical anterior disc displacement.

This study has characterized a rat model with temporomandibular osteoarthritis (TMJ-OA) following a surgical anterior displacement of their articular disc (ADD). The well-established model of OA, induced by an intra-articular injection of complete Freund's adjuvant (CFA) into the TMJ, was used for comparison purposes. Male Wistar rats were assigned into two surgical groups, namely, ADD (anterior disc displacement) and sham-operated (surgical access, without ADD). Additional groups received an intra-articular infiltration of CFA (50 µl/site; 1:1 oil/saline emulsion), or the vehicle (0.9% NaCl). The separate experimental subgroups were euthanized at 15, 30 or 60 days and their left TMJs were collected for histological, immunohistochemistry and micro-CT analyses. The serum levels of IL-1ß, IL-6 and TNF were analyzed. The fibrocartilage thicknesses were increased in the ADD groups at all of the analyzed time-points. In the CFA group, fibrocartilage thickenings were seen only in the posterior thirds at 15 days. The ADD group displayed an increase of the proteoglycan contents and ADAMTS5 immunopositivity in the fibrocartilage at 30 and 60 days, without any variations of the collagen contents or the osteoclast activation. Upon the micro-CT evaluation, the ADD group presented increments of their trabecular separations and bone surfaces, with reduced trabecular thicknesses and bone volumes, plus osteophyte formations and condyle flattenings, from 30 to 60 days. The IL-1ß, TNF or IL-6 serum levels were undetectable. The surgical ADD in the rats led to long-term OA-like alterations, with typical structural and morphological derangements of the TMJ, representing a reliable experimental model to investigate the TMJ-OA-related mechanisms.

Toxicological profile of IQG-607 after single and repeated oral administration in minipigs: An essential step towards phase I clinical trial.

IQG-607 is an anti-tuberculosis drug candidate, with a promising safety and efficacy profile in models of tuberculosis infection both in vitro and in vivo. Here, we evaluated the safety and the possible toxic effects of IQG-607 after acute and 90-day repeated administrations in minipigs. Single oral administration of IQG-607 (220 mg/kg) to female and male minipigs did not result in any morbidity or mortality. No gross lesions were observed in the minipigs at necropsy. Repeated administration of IQG 607 (65, 30, or 15 mg/kg), given orally, for 90 days, in both male and female animals did not cause any mortality and no significant body mass alteration. Diarrhea and alopecia were the clinical signs observed in animals dosed with IQG-607 for 90 days. Long-term treatment with IQG-607 did not induce evident alterations of blood cell counts or any hematological parameters. Importantly, the repeated schedule of administration of IQG-607 resulted in increased cholesterol levels, increased glucose levels, decrease in the globulin levels, and increased creatinine levels over the time. Most necropsy and histopathological alterations of the organs from IQG-607-treated groups were also observed for the untreated group. In addition, pharmacokinetic parameters were evaluated. IQG-607 represents a potential candidate molecule for anti-tuberculosis drug development programs. Its promising in vivo activity and mild to moderate toxic events detected in this study suggest that IQG-607 represents a candidate for clinical development.

Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages.

INTRODUCTION: The safety of orthodontic materials is a matter of high interest. In this study, we aimed to assess the in-vitro cytotoxicity of orthodontic band extracts, with and without silver solder, by comparing the viability outcomes of the HaCat keratinocytes, the fibroblastic cell lineages HGF and MRC-5, and the kidney epithelial Vero cells. METHODS: Sterilized orthodontic bands with and without silver solder joints were added to culture media (6 cm2/mL) and incubated for 24 hours at 37°C under continuous agitation. Subsequently, the cell cultures were exposed to the obtained extracts for 24 hours, and an assay was performed to evaluate the cell viability. Copper strip extracts were used as positive control devices. RESULTS: The extracts from orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF was not altered by this material. Conversely, the extracts of orthodontic bands without silver solder did not significantly modify the viability index of all evaluated cell lines. CONCLUSIONS: Except for HGF fibroblasts, all tested cell lines showed decreased viability percentages after exposure to extracts of orthodontic bands containing silver solder joints. These data show the relevance of testing the toxicity of orthodontic devices in different cell lines.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

BACKGROUND Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton’s medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton’s medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.

Effects of caffeine on behavioral and inflammatory changes elicited by copper in zebrafish larvae: Role of adenosine receptors.

This study investigated the effects of caffeine in the behavioral and inflammatory alterations caused by copper in zebrafish larvae, attempting to correlate these changes with the modulation of adenosine receptors. To perform a survival curve, 7dpf larvae were exposed to 10µM CuSO4, combined to different concentrations of caffeine (100µM, 500µM and 1mM) for up to 24h. The treatment with copper showed lower survival rates only when combined with 500µM and 1mM of caffeine. We selected 4 and 24h as treatment time-points. The behavior evaluation was done by analyzing the traveled distance, the number of entries in the center, and the length of permanence in the center and the periphery of the well. The exposure to 10µM CuSO4 plus 500µM caffeine at 4 and 24h changed the behavioral parameters. To study the inflammatory effects of caffeine, we assessed the PGE2 levels by using UHPLC-MS/MS, and TNF, COX-2, IL-6 and IL-10 gene expression by RT-qPCR. The expression of adenosine receptors was also evaluated with RT-qPCR. When combined to copper, caffeine altered inflammatory markers depending on the time of exposure. Adenosine receptors expression was significantly increased, especially after 4h exposure to copper and caffeine together or separately. Our results demonstrated that caffeine enhances the inflammation induced by copper by decreasing animal survival, altering inflammatory markers and promoting behavioral changes in zebrafish larvae. We also conclude that alterations in adenosine receptors are related to those effects.

Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis. The better understanding of important metabolic pathways from M. tuberculosis can contribute to the development of novel therapeutic and prophylactic strategies to combat TB. Nucleoside hydrolase (MtIAGU-NH), encoded by iunH gene (Rv3393), is an enzyme from purine salvage pathway in M. tuberculosis. MtIAGU-NH accepts inosine, adenosine, guanosine, and uridine as substrates, which may point to a pivotal metabolic role. OBJECTIVES: Our aim was to construct a M. tuberculosis knockout strain for iunH gene, to evaluate in vitro growth and the effect of iunH deletion in M. tuberculosis in non-activated and activated macrophages models of infection. METHODS: A M. tuberculosis knockout strain for iunH gene was obtained by allelic replacement, using pPR27xylE plasmid. The complemented strain was constructed by the transformation of the knockout strain with pNIP40::iunH. MtIAGU-NH expression was analysed by Western blot and LC-MS/MS. In vitro growth was evaluated in Sauton's medium. Bacterial load of non-activated and interferon-γ activated RAW 264.7 cells infected with knockout strain was compared with wild-type and complemented strains. FINDINGS: Western blot and LC-MS/MS validated iunH deletion at protein level. The iunH knockout led to a delay in M. tuberculosis growth kinetics in Sauton's medium during log phase, but did not affect bases and nucleosides pool in vitro. No significant difference in bacterial load of knockout strain was observed when compared with both wild-type and complemented strains after infection of non-activated and interferon-γ activated RAW 264.7 cells. MAIN CONCLUSION: The disruption of iunH gene does not influence M. tuberculosis growth in both non-activated and activated RAW 264.7 cells, which show that iunH gene is not important for macrophage invasion and virulence. Our results indicated that MtIAGU-NH is not a target for drug development.