The Cytokine IL-1ß and Piperine Complex Surveyed by Experimental and Computational Molecular Biophysics.

The bioactive piperine, a compound found in some pepper species, has been widely studied because of its therapeutic properties that include the inhibition of an important inflammation pathway triggered by interleukin-1 beta (IL-1ß). However, investigation into the molecular interactions between IL-1ß and piperine is not reported in the literature. Here, we present for the first time the characterisation of the complex formed by IL-1ß and piperine through experimental and computational molecular biophysical analyses. Fluorescence spectroscopy unveiled the presence of one binding site for piperine with an affinity constant of 14.3 × 104 M-1 at 298 K. The thermodynamic analysis indicated that the interaction with IL-1ß was spontaneous (∆G = -25 kJ/mol) and, when split into enthalpic and entropic contributions, the latter was more significant. Circular dichroism spectroscopy showed that piperine did not affect IL-1ß secondary structure (~2%) and therefore its stability. The set of experimental data parameterized the computational biophysical approach. Through molecular docking, the binding site micro-environment was revealed to be composed mostly by non-polar amino acids. Furthermore, molecular dynamics, along with umbrella sampling, are in agreement with the thermodynamic parameters obtained by fluorescence assays and showed that large protein movements are not present in IL-1ß, corroborating the circular dichroism data.

Silencing the Oryza sativa plasma membrane H+-ATPase isoform OsA2 affects grain yield and shoot growth and decreases nitrogen concentration.

The plasma membrane (PM) H+-ATPase (EC 3.6.1.3.) is a key component involved in nutrient uptake. There are 10 PM H+-ATPase isoforms in the rice genome (OsA1-OsA10), and OsA2 is highly responsive to nitrate (NO3-). We investigated the role that the OsA2 isoform plays in the total N and growth of rice (Oryza sativa). By the use of artificial microRNA, mutant osa2 rice lines presented ∼70 % downregulated levels of OsA2. Three osa2 lines and control plants (transformed with an empty IRS154 vector and named IRS) were cultivated in the greenhouse to evaluate grain and shoot production. For hydroponic experiments, the same lines were grown in Hoagland solution under two different NO3- levels for 30 days - 0.2 mM NO3--N (low N) or 2.0 mM NO3--N (sufficient N) - or were grown for three days without NO3- (starvation) after 27 days under 2.0 mM NO3--N. In the greenhouse experiments, compared with the IRS plants, the osa2 lines had lower shoot fresh weights, grain yields and SPAD values. Moreover, compared with the IRS plants, the three osa2 lines grown hydroponically under low NO3- levels had lower N concentration and net flux of NO3-. PM H+-ATPase activity was lower in the osa2 mutants than in the IRS plants. The relatively low N concentration in the osa2 lines was not due to lower expression of OsNRT2.1, OsNRT2.2, or OsNAR2.1. These results indicate that the specific PM H+-ATPase isoform OsA2 affects the net flux of NO3-, N concentration, and grain yield.

Details of the cooperative binding of piperlongumine with rat serum albumin obtained by spectroscopic and computational analyses.

Piperlongumine (PPL) has presented a variety of important pharmacological activities. In recent pharmacokinetics studies in rats, this molecule reached 76.39% of bioavailability. Although PPL is present in the bloodstream, no information is found on the interaction between PPL and rat serum albumin (RSA), the most abundant protein with the function of transporting endo/exogenous molecules. In this sense, the present study elucidated the mechanism of interaction between PPL and RSA, using in conjunction spectroscopic and computational techniques. This paper shows the importance of applying inner filter correction over the entire fluorescence spectrum prior to any conclusion regarding changes in the polarity of the fluorophore microenvironment, also demonstrates the convergence of the results obtained from the treatment of fluorescence data using the area below the spectrum curve and the intensity in a single wavelength. Thermodynamic parameters revealed that PPL binds to RSA spontaneously (ΔG < 0) and the process is entropically driven. Interaction density function method (IDF) indicated that PPL accessed two cooperative sites in RSA, with moderate binding constants (2.3 × 105 M-1 and 1.3 × 105 M-1). The molecular docking described the microenvironment of the interaction sites, rich in apolar residues. The stability of the RSA-PPL complex was checked by molecular dynamics.

Mussels (Perna perna) as bioindicator of environmental contamination by Cryptosporidium species with zoonotic potential.

Sources of contamination such as animal feces runoff, organic fertilizer application, and the release of partially treated or untreated sewage can lead to the contamination of aquatic environments by Cryptosporidium spp. The quality of mussels as food is closely related to the sanitary conditions of the marine environment where these bivalves are found. Marine mollusks are filter feeders that are able to retain Cryptosporidium oocysts in their tissue, thus functioning as bioindicators. A total of 72 pooled mussel samples of the species Perna perna were collected at two sites (A and B) in the municipality of Mangaratiba, Rio de Janeiro State, Brazil. Sampling involved removal of 30 mussels, from each collection site every month for one year. The 30 mussels from each sampling were then allocated into three groups of 10. Two Cryptosporidium spp. genes (18S and GP60) were targeted for DNA amplification from the samples obtained. After purification, all of the products obtained were sequenced and phylogenetic analyses were performed. Of the 72 samples analyzed using the nested-PCR for the 18S gene target, 29.2% were positive for the presence of Cryptosporidium spp. Of these samples, 52.4% were collected at site A (ie 11/21) and 47.6% at site B (ie 10/21). The 18S genes of all the samples considered positive for Cryptosporidium spp. were sequenced, and the following three species were identified: Cryptosporidium parvum, C. meleagridis, and C. andersoni. Three distinct C. parvum subtypes (IIaA19G2R2; IIaA20G2R2; IIaA20G3R2) were identified using the GP60 gene. More studies to evaluate the zoonotic potential of this species should be performed as both sampling locations contain human and/or animal fecal contaminants.

Overexpression of the anaphase-promoting complex (APC) genes in Nicotiana tabacum promotes increasing biomass accumulation.

The anaphase-promoting complex (APC) plays pivotal roles in cell cycle pathways related to plant development. In this study, we present evidence that overproduction of APC10 from Arabidopsis thaliana in tobacco (Nicotiana tabacum) plants promotes significant increases in biomass. Analyzes of plant's fresh and dried weight, root length, number of days to flower and number of seeds of plants overexpressing AtAPC10 verified an improved agronomic performance of the transgenic plants. Detailed analyzes of the leaf growth at the cellular level, and measurements of leaf cell number, showed that AtAPC10 also produce more cells, showing an enhancement of proliferation in these plants. In addition, crossing of plants overexpressing AtAPC10 and AtCDC27a resulted in a synergistic accumulation of biomass and these transgenic plants exhibited superior characteristics compared to the parental lines. The results of the present study suggest that transgenic plants expressing AtAPC10 and AtAPC10/AtCDC27a concomitantly are promising leads to develop plants with higher biomass.

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development.

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.