Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.     

Versican G3 promotes mouse mammary tumor cell growth, migration, and metastasis by influencing EGF receptor signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.     

T Cell Activation and Cytokine Profile of Tuberculosis and HIV-Positive Individuals during Antituberculous Treatment and Efavirenz-Based Regimens

Introduction

The profile of immune activation markers in tuberculosis and HIV-infected patients is already known. The impact of simultaneous infections on the immune parameters is still not fully explored.

Methods

We conducted a prospective study to estimate trajectories of activated T cell subsets and the profile of anti- and pro-inflammatory cytokines in a group of HIV-TB individuals, previously naïve for HAART, recruited from a randomized clinical trial during TB treatment and first antiretroviral therapy with efavirenz. Patients were evaluated according to the immunosuppression levels at baseline as group 1 (CD4<200 cells/mm³) and group 2 (CD4>200 cells/mm³). These parameters were measured at the time of HAART initiation (started about 30 days after the onset of TB treatment) and at the follow-up visits after 30, 60, 90 and 180 days. Trajectories were estimated using least squares estimates of the coefficients of a restricted cubic spline function in time after adjusting for subject effects, bootstrapping it 500 times.

Results

Increase of CD4 T cell counts and suppression of HIV viral load were observed for all patients under HAART and TB treatment. Descendent trajectories were observed for the activated CD8⁺/CD38⁺ and CD3⁺/HLA-DR⁺ T cell subsets, and for plasma concentration of gamma- interferon (IFN-γ). Except for TNF-α and IL-2 discrete variations were observed for the other cytokines. Differences in the trajectories of these parameters were observed for groups 1 and 2. Higher values of IFN-γ, IL-2, IL-6 and IL-10 were observed for group 1 from the baseline to two months after treatment initiation, whereas reduced levels of TNF-α were observed for this group between 60 and 120 days of HAART.

Conclusion

Independent of the immunosuppression profile at baseline, HIV-TB patients under HAART were able to recover the CD4⁺ T cell counts, and control viral replication and immune activation parameters over time.     

Label-Free Enrichment of Adrenal Cortical Progenitor Cells Using Inertial Microfluidics

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin contamination in <Emphasis Type="Italic">Bt</Emphasis> and non-<Emphasis Type="Italic">Bt</Emphasis> maize cultivated in Brazil

Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Cellular membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves C3b, an essential component of the complement system

Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.