Reference genes for normalization: a study of rat brain tissue

Quantitative real-time polymerase chain reaction (qPCR) has become a widely used tool in the search for disease genes. When examining gene expression with qPCR in psychiatric diseases, endogenous reference gene(s) must be used for normalization. Traditionally, genes such as beta-actin (ActB), Gapd, and 18s rRNA, assumed to be stably expressed, have been used for normalization. However, it has become clear that expression of these genes is influenced by different experimental paradigms. Since stable gene expression of houskeeping genes (HKGs), therefore, cannot be expected, alternative strategies are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and geNorm software applets. Additionally, we have applied four different normalization approaches for normalization of the three target genes. We found using the NormFinder software that Ywhaz is the most stably expressed gene among the eight tested HKGs. However, the results of the analysis of the target genes are highly dependent on the choice of normalization approach. Moreover, the number of HKGs, used for selection of the most stable HKG, also influences on the result of the gene expression analysis of target genes. These results underline the importance of choosing a proper normalization strategy when analyzing gene expression with qPCR. The strategy should be unbiased and carried out in every specific experimental setting.     

The impact of antiretroviral therapy and isoniazid preventive therapy on tuberculosis incidence in HIV-infected patients in Rio de Janeiro, Brazil

BACKGROUND: Tuberculosis is a common complication and leading cause of death in HIV infection. Antiretroviral therapy (ART) lowers the risk of tuberculosis, but may not be sufficient to control HIV-related tuberculosis. Isoniazid preventive therapy (IPT) reduces tuberculosis incidence significantly, but is not widely used. METHODS: We analysed tuberculosis incidence in 11 026 HIV-infected patients receiving medical care at 29 public clinics in Rio de Janeiro, Brazil, between 1 September 2003 and 1 September 2005. Data were collected through a retrospective medical record review. We determined rates of tuberculosis in patients who received neither ART nor IPT, only ART, only IPT, or both ART and IPT. RESULTS: The overall tuberculosis incidence was 2.28 cases/100 person-years (PY) [95% confidence interval (CI) 2.06-2.52]. Among patients who received neither ART nor IPT, incidence was 4.01/100 PY. Patients who received ART had an incidence of 1.90/100 PY (95% CI 1.66-2.17) and those treated with IPT had a rate of 1.27/100 PY (95% CI 0.41-2.95). The incidence among patients who received ART and IPT was 0.80/100 PY (95% CI 0.38-1.47). Multivariate Cox proportional hazards modeling revealed a 76% reduction in tuberculosis risk among patients receiving both ART and IPT (adjusted relative hazard 0.24; P < 0.001) after adjusting for age, previous tuberculosis diagnosis, and CD4 cell counts at baseline. CONCLUSION: The use of both IPT and ART in HIV-infected patients is associated with significantly reduced tuberculosis incidence. In conjunction with expanded access to ART, the wider use of IPT in patients with HIV will improve tuberculosis control in high burden areas.     

Multiple antibiotic-resistance mechanisms including a novel combination of extended-spectrum beta-lactamases in a Klebsiella pneumoniae clinical strain isolated in Argentina

Klebsiella pneumoniae M1803, isolated from a paediatric patient with chronic urinary infection, presented nine antimicrobial resistance mechanisms harboured on two conjugative megaplasmids, in addition to the chromosomally mediated SHV-1 beta-lactamase. These nine antimicrobial resistance mechanisms comprised two extended-spectrum beta-lactamases (ESBLs) (PER-2 and CTX-M-2), TEM-1-like, OXA-9-like, AAC(3)-IIa, AAC(6')-Ib, ANT(3")-Ia and resistance determinants to tetracycline and chloramphenicol. During fluoroquinolone treatment, a variant derived from M1803 (named M1826) was selected, with an overall increase of MICs, in particular of cefoxitin and carbapenems. No enzymic activity against these latter drugs was found. Mutations in the region analogous to the quinolone resistance-determining region were not found. Strain M1826 was deficient in OmpK35/36 expression, which produced the decrease in the susceptibility to cefoxitin, carbapenems and fluoroquinolones. The blaCTX-M-2 gene was located in an unusual class 1 integron, which includes Orf513, as occurred in the recently described In35. In addition, Tn3 and Tn1331 were detected in both K. pneumoniae isolates. This is the first report of in vivo selection of an OmpK35/36 deficiency in a K. pneumoniae strain that produced a novel combination of two ESBLs (CTX-M-2 and PER-2) during fluoroquinolone treatment in a paediatric patient with chronic urinary infection.     

HLA study on two Mexican Mestizo families with autoimmune thyroid disease

Several studies have been done regarding the genetic susceptibility to autoimmune thyroid disease, particularly those related to the role of Major Histocompatibility Complex (MHC) genes in the etiology of the disease. In the present study, we report class I and class II MHC haplotypes in nine individuals affected by Hashimoto thyroiditis and Graves' disease who belong to two distinct Mexican families. In one of the families, Hashimoto thyroiditis was associated with the Human Leukocyte Antigen (HLA) HLA-DR3 allele whereas in the other family the disease was associated with homozygosity for the HLA-DR4 (DRB1*0407), HLA-DQ3 (DQB1*0302) haplotype. On the other hand, Graves' disease was found to be associated in one of the families with HLA-DR2 (DRB1*1501) and in the other with homozygosity for the HLA-DR7 (DRB*0701) and HLA-DQ2 (DQB1*0201) haplotype. These results confirm that in Mexicans as in other ethnic groups, genes located within the MHC region are related to the genetic susceptibility to develop autoimmune thyroid disease.     

Ligand-free Pd/Ag-mediated dehydrogenative alkynylation of imidazole derivatives

A variety of 2-alkynyl(benzo)imidazoles have been synthesized by dehydrogenative alkynylation of (benzo)imidazoles with terminal alkyne in NMP under air in the presence of Ag₂CO₃ as the oxidant and Pd(OAc)₂ as the catalyst precursor. The data obtained in this study support a reaction mechanism involving a non-concerted metalation deprotonation (n-CMD) pathway.

The regioselective synthesis of 2-alkynyl(benz)imidazoles was successfully achieved by Pd(ii)/Ag(i)-mediated dehydrogenative alkynylation of the corresponding (benz)imidazoles with terminal alkynes in an open vessel.

The development of synthetic protocols that enable the direct and selective functionalization of C_sp²–H bonds are of primary importance in the decoration of the imidazole scaffold.^1–10 The presence of two nitrogen atoms in the pentatomic structure of this important azole in fact introduces a differentiation in the chemical behaviour of the three C–H bonds, allowing their selective involvement in carbon–carbon bond-forming reactions as long as appropriate experimental conditions are identified.During our studies dedicated to the synthesis and investigation of azole-based fluorophores featuring a π-conjugated backbone end-capped with electron-donating (EDG) and electron-acceptor (EWG) groups (the so-called “push–pull” systems),^11–15 taking into account the structural characteristics and synthetic versatility of a triple carbon–carbon bond^16–23 that make it an excellent π-spacer, we recently decided to evaluate the possibility of obtaining alkynyl-substituted imidazoles by transition metal-catalyzed C_sp²–C_sp bond-forming reactions. Among the synthetic procedures that allow the selective alkynylation of imidazoles and other five-membered heteroarenes,^5,24 there is no doubt that the possibility of forming the new σ carbon–carbon bond through the double activation of two distinct carbon–hydrogen bonds through a cross-dehydrogenative alkynylation (CDA) represents the best synthetic approach in terms of atom economy and functional group tolerance (eqn (1), Scheme 1).^25–34 In fact, when compared with the transition metal-catalyzed direct C_sp²–H alkynylation protocols involving 1-haloalkynes (the so-called “inverse Sonogashira coupling”) (eqn (2), Scheme 1),^35–45 hypervalent iodine reagents (eqn (3), Scheme 1),^46–48 or α,β-ynoic acids (decarboxylative direct arylation) (eqn (4), Scheme 1),^26,49,50 the CDA methodology makes it possible to use terminal alkynes without the need for preliminary activation.^5,24Open in a separate windowScheme 1Synthetic protocols for the transition metal-catalyzed C_sp²–H alkynylation of heteroarenes.Despite several papers having been dedicated to the dehydrogenative alkynylation of pyrroles,²⁷ indoles,^27,31 oxazoles,^{27,30,32–34} benzoxazoles,^{26,29,30,32,34} thiazoles,^27,30 benzothiazoles,^{26,29,31,32,34} pyrazoles,^25,27 1,3,4-oxadiazoles,³³ imidazopyridines,^27,34 and N-substituted sydnones,²⁸ to the best of our knowledge a study specifically devoted to the dehydrogenative alkynylation of imidazoles has never been reported. However, careful reading the literature where alkynylation reactions involving azoles other than imidazole were described, it emerged that two papers reported the C-2 alkynylation of N-benzylimidazole and N-benzylbenzimidazole with phenylacetylene. In a study mainly devoted to the dehydrogenative alkynylation of imidazo[1,5-a]pyridine, Shihabara, Dohke and Murai employed the commercially unavailable Pd(ii) complex bi(4-nitropyridinyl)Pd(OAc)₂, Ag₂CO₃ as the oxidant and AcOH as additive, in a 95 : 5 mixture of DMF and DMSO for 2 h at 120 °C under argon.³⁴ Due to the propensity of alkynes to give Glaser-type homocoupling side-products in the presence of silver(i) salts,⁵¹ phenylacetylene was slowly added over 90 min to the reaction mixture. In 2015, Likhar, Kantam and co-workers used a synthetic Pd(ii) carbene complex. In this case Ag₂O was used as the oxidant, Cs₂CO₃ base, performing the coupling in DMF at 85 °C under air.²⁶Encouraged by these results but with the intention of developing a simplified procedure that would allow the use of a commercial Pd(ii) pre-catalyst in the absence of any palladium ligands, we decided to review the conditions of reaction and, in this work, we are pleased to summarize the results obtained in the synthesis of 2-alkynylimidazole derivatives by dehydrogenative alkynylation with terminal alkynes (Scheme 2). In particular, a careful screening allowed us to show how the reactivity of C_sp²–H bond of imidazole derivatives and other azoles can be enhanced simply by performing the alkynylation using NMP as the reaction solvent, under air in non-anhydrous conditions, and without the need for palladium ligands.Open in a separate windowScheme 2Pd/Ag-Promoted dehydrogenative alkynylation of imidazole derivatives.We decided to start our synthetic investigations by trying a cross-dehydrogenative coupling between N-methylbenzimidazole (1) and phenylacetylene (2a), chosen as typical coupling partners, under conditions very similar to those proposed by Murai and co-workers for the regioselective C-3 dehydrogenative alkynylation of 1-alkynyl-3-arylimidazo[1,5-a]-pyridines.³⁴Hence, to a mixture of 1.0 mmol 1, 5 mol% Pd(OAc)₂, 1.5 equiv. Ag₂CO₃, and 1.0 equiv. AcOH in a 95 : 5 (v/v) mixture of DMF and DMSO under argon 3.0 equiv. of 2a were added dropwise over 90 min. However, after 2 h at 120 °C a 56% GLC conversion of 1 was recorded, and the required 2-phenylethynyl-N-methylbenzimidazole (3a) was obtained in only 34% GLC yield (entry 1,

A Long-Term Energy-Rich Diet Increases Prefrontal BDNF in Sprague-Dawley Rats

Findings of the effect of high-fat feeding including “Cafeteria Diets” (CAF) on brain-derived neurotrophic factor (BDNF) in the hippocampus (HIP) and prefrontal cortex (PFC) in rodents are conflicting. CAF is a non-standardized, highly palatable energy-rich diet composed by everyday food items for human consumption and is known to induce metabolic syndrome and obesity in rats. However, the highly palatable nature of CAF may counteract a negative effect of chronic stress on anticipatory behavior and synaptic plasticity in the hippocampus, hence represent a confounding factor (e.g., when evaluating functional effects on the brain). This study investigated the effects of a chronic, restricted access to CAF on BDNF, monoamine neurotransmitters, and redox imbalance in HIP and PFC in male rats. Our results show that CAF induced BDNF and its receptor TrkB in PFC compared to the controls (p < 0.0005). No differences in monoamine neurotransmitters were detected in either PFC or HIP. CAF increased dehydroascorbic acid and decreased malondialdehyde in PFC (p < 0.05), suggesting an early redox imbalance insufficient to induce lipid peroxidation. This study supports that a chronic CAF on a restricted schedule increases BDNF levels in the PFC of rats, highlighting that this may be a suboptimal feeding regime when investigating the effects of diet-induced obesity in the brain and emphasizing this as a point of attention when comparing the findings.     

Human mAChR antibodies from Sjögren syndrome sera increase cerebral nitric oxide synthase activity and nitric oxide synthase mRNA level

We demonstrated the presence of circulating antibodies from Sjögren syndrome (SS) patients enable to interact with rat cerebral frontal cortex by activating muscarinic acetylcholine receptors (mAChRs). IgG from SS and IgG from normal subjects were studied by flow cytometry, enzyme immunoassay (ELISA), and radioligand binding assays. By flow cytometric and ELISA procedures, it was shown that IgG from SS patients reacted to cerebral frontal cortex cell surface. SS IgG was able to interact with mAChR by inhibiting ³H-QNB binding to its specific receptor. Besides, SS IgG displayed an agonistic-like activity associated to specific M₁ and M₃ mAChR activation, increasing nitric oxide synthase (NOS) isoform activities. Neuronal (n) and endothelial (e) NOS-mRNA gene expression of rat frontal cortex is induced by SS IgG. This article supports the participation of humoral immune alterations in SS, resulting in central parasympathetic functional deregulation. These antibodies alter mAChR activation, NOS activity, and eNOS and nNOS gene expression.     

Brazilian patients with panic disorder: the use of defense mechanisms and their association with severity

This study aims to evaluate the defense mechanisms most frequently used by Brazilian patients with panic disorder when compared with a control group. The study also examines the association between severity of disease and comorbidity and the use of specific defense mechanisms. Sixty panic-disordered patients and 31 controls participated in the study. The Mini International Neuropsychiatric Interview was used to confirm the panic disorder diagnosis and to establish the comorbid diagnosis. The Clinical Global Impression (CGI) was used to assess severity and the Defensive Style Questionnaire (DSQ-40) was used to evaluate the defense mechanisms. Panic patients used more neurotic (mean = 4.9 versus 3.6; p < 0.001) and immature (mean = 3.9 versus 2.8; p < 0.001) defenses as compared with controls. Panic patients with severe disease (n = 37; CGI>4) had more depression comorbidity and used more immature defenses than patients with CGI相似文献