Coxiella burnetii, the agent of Q fever in Brazil: its hidden role in seronegative arthritis and the importance of molecular diagnosis based on the repetitive element IS1111 associated with the transposase gene

Coxiella burnetii is the agent of Q fever , an emergent worldwide zoonosis of wide clinical spectrum. Although C. burnetii infection is typically associated with acute infection, atypical pneumonia and flu-like symptoms, endocarditis, osteoarticular manifestations and severe disease are possible, especially when the patient has a suppressed immune system; however, these severe complications are typically neglected. This study reports the sequencing of the repetitive element IS1111 of the transposase gene of C. burnetii from blood and bronchoalveolar lavage (BAL) samples from a patient with severe pneumonia following methotrexate therapy, resulting in the molecular diagnosis of Q fever in a patient who had been diagnosed with active seronegative polyarthritis two years earlier. To the best of our knowledge, this represents the first documented case of the isolation of C. burnetii DNA from a BAL sample.     

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)?? = 2 μM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC?? = 8 μM) and La10 (IC?? = 10 μM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.     

Antiviral therapy against chronic hepatitis B in Brazil: high rates of lamivudine resistance mutations and correlation with HBV genotypes

The effectiveness of antiviral treatments of chronic hepatitis B has been poorly studied in Brazil. Here, hepatitis B virus (HBV) DNA positivity, drug resistance mutations and their association with HBV genotypes were evaluated in chronically HBV-infected patients under different drug regimens in Brazil. The study involved 129 patients under interferon or nucleos(t)ide analogue therapy for a median treatment time of 12 months. One hundred and five (81%) of these patients were treated with lamivudine (LAM), either in monotherapy or in combination with newer drugs, such as entecavir (ETV) or tenofovir (TDF). High (37.5-100%) rates of HBV DNA positivity were observed with all but one drug regimen (LAM + ETV). However, patients that were treated with ETV alone, TDF alone or with LAM combination therapies had a mean viral load that was 3-4 log lower than patients treated with LAM monotherapy. Of the patients treated with LAM, 47% developed resistance mutations. HBV genotypes A (59.1%), D (30.3%) and F (9.1%) were found. There was no association between the presence of LAM resistance mutations and genotypes, HBeAg status or treatment duration. Nevertheless, the rtM204V mutation was observed more frequently (12/13, 92%) in genotype A than in the others (p = 0.023). Six out of nine isolates that contained the rtM204I mutation belonged to genotype D and half of them displayed a single mutation. Genotype D isolates with the rtM204V variant preferentially displayed a triple mutation, while genotype A preferentially displayed a double mutation (p = 0.04).     

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.     

Purification and Biological Activities of Abelmoschus esculentus Seed Lectin

The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0?kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1?mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44?%, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10?mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69?%, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0?kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.     

Molecular interactions of c-ABL mutants in complex with imatinib/nilotinib: a computational study using linear interaction energy (LIE) calculations

In spite of the effectiveness of Imatinib for chronic myeloid leukemia (CML) treatment, resistance has repeatedly been reported and is associated with point mutations in the BCR-ABL chimeric gene. To overcome this resistance, several inhibitors of BCR-ABL tyrosine kinase activity were developed. In this context, computational simulations have become a powerful tool for understanding drug-protein interactions. Herein, we report a comparative molecular dynamics analysis of the interaction between two tyrosine kinase inhibitors (imatinib or nilotinib) against wild type c-ABL protein and 12 mutants, using the semi-empirical linear interaction energy (LIE) method, to assess the feasibility of this approach for studying resistance against the inhibitory activity of these drugs. In addition, to understand the structural changes that are associated with resistance, we describe the behavior of water molecules that interact simultaneously with specific residues (Glu286, Lys271 and Asp381) of c-ABL (wild type or mutant) and their relationship with drug resistance. Experimental IC50 values for the interaction between imatinib, wild type c-ABL, and 12 mutants were used to obtain the proper LIE coefficients (α, β and γ) to estimate the free energy of the binding of imatinib with wild-type and mutant proteins, and values were extrapolated for the analysis of the nilotinib/c-ABL interaction. Our results indicate that LIE was suitable to predict the superior inhibitory activity of nilotinib and the resistance to inhibition that was observed in c-ABL mutants. Additionally, for c-ABL mutants, the observed number of water molecules being turned over while interacting with amino acids Glu286, Lys271 and Asp381 was associated with resistance to imatinib, resulting in a less effective inhibition of the kinase activity.