Evolution of Cyclic Amidohydrolases: A Highly Diversified Superfamily

Dihydroorotases are universal proteins catalyzing the third step of pyrimidine biosynthesis. These zinc metalloenzymes belong to the superfamily of cyclic amidohydrolases, comprising also other enzymes that are involved in degradation of either purines (allantoinases), pyrimidines (dihydropyrimidinases) or hydantoins (hydantoinases). The evolutionary relationships between these mechanistically related enzymes were estimated after designing a method to build an accurate multiple sequence alignment. The amino acid sequences that have been crystallized were used to build a seed alignment. All the remaining homologues were progressively added by aligning their HMM profiles to the seed HMM profile, allowing to obtain a reliable phylogeny of the superfamily. This helped us to propose a new evolutionary classification of dihydroorotases into three major types, while at the same time disentangling an important part of the history of their complex structure–function relationships. Although differing in their substrate specificity, allantoinases, hydantoinases and dihydropyrimidinases are found to be phylogenetically closer to DHOase Type I than the proximity of the three DHOase types to each other. This suggests that the primordial cyclic amidohydrolase was a multifunctional, highly evolvable generalist, with high conformational diversity allowing for promiscuous activities. Then, successive gene duplications allowed resolving the primordial substrate ambiguity in various substrate specificities. The present-day superfamily of cyclic amidohydrolases is the result of the progressive divergence of these ancestral paralogous copies by descent with modification.     

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.     

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration.     

New records of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) predation by Brazilian Hemipteran predatory bugs

The tomato borer Tuta absoluta, native to western South America, is an extremely devastating pest in tomato crops in most of South America, Europe and Africa North of the Sahel, causes yield losses up to 100% and decreases fruit quality in open field and greenhouse crops if control methods are not applied. In Brazil two other important lepidopteran pests – Neoleucinodes elegantalis and Helicoverpa zea – occur in tomato, as well as thrips, whiteflies and aphids. For control of these pests, frequent applications of pesticides of up to 5 times per week are needed, and these resulted in the appearance of resistant populations to a number of active ingredients and decimation of natural enemies. Biological control may offer a better, safer and more sustainable opportunity for pest management. Mirid predatory bugs are currently used with success in southern Europe to control T. absoluta and other pests. In Brazil, four Hemipteran predatory bugs, not yet known to attack T. absoluta, were found to successfully prey on eggs and larvae of this pest. The first results on their predation capacity, development, survival and reproduction on T. absoluta on tomato plants are presented.     

Distinguishing Antimicrobial Models with Different Resistance Mechanisms via Population Pharmacodynamic Modeling

Semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) modeling is increasingly used for antimicrobial drug development and optimization of dosage regimens, but systematic simulation-estimation studies to distinguish between competing PD models are lacking. This study compared the ability of static and dynamic in vitro infection models to distinguish between models with different resistance mechanisms and support accurate and precise parameter estimation. Monte Carlo simulations (MCS) were performed for models with one susceptible bacterial population without (M1) or with a resting stage (M2), a one population model with adaptive resistance (M5), models with pre-existing susceptible and resistant populations without (M3) or with (M4) inter-conversion, and a model with two pre-existing populations with adaptive resistance (M6). For each model, 200 datasets of the total bacterial population were simulated over 24h using static antibiotic concentrations (256-fold concentration range) or over 48h under dynamic conditions (dosing every 12h; elimination half-life: 1h). Twelve-hundred random datasets (each containing 20 curves for static or four curves for dynamic conditions) were generated by bootstrapping. Each dataset was estimated by all six models via population PD modeling to compare bias and precision. For M1 and M3, most parameter estimates were unbiased (<10%) and had good imprecision (<30%). However, parameters for adaptive resistance and inter-conversion for M2, M4, M5 and M6 had poor bias and large imprecision under static and dynamic conditions. For datasets that only contained viable counts of the total population, common statistical criteria and diagnostic plots did not support sound identification of the true resistance mechanism. Therefore, it seems advisable to quantify resistant bacteria and characterize their MICs and resistance mechanisms to support extended simulations and translate from in vitro experiments to animal infection models and ultimately patients.     

Effects of MDMA Injections on the Behavior of Socially-Housed Long-Tailed Macaques (Macaca fascicularis)

3,4-methylenedioxy-N-methyl amphetamine (MDMA) is one of the few known molecules to increase human and rodent prosocial behaviors. However, this effect has never been assessed on the social behavior of non-human primates. In our study, we subcutaneously injected three different doses of MDMA (1.0, 1.5 or 2.0mg/kg) to a group of three, socially housed, young male long-tailed macaques. More than 200 hours of behavioral data were recorded, during 68 behavioral sessions, by an automatic color-based video device that tracked the 3D positions of each animal and of a toy. This data was then categorized into 5 exclusive behaviors (resting, locomotion, foraging, social contact and object play). In addition, received and given social grooming was manually scored. Results show several significant dose-dependent behavioral effects. At 1.5mg/kg only, MDMA induces a significant increase in social grooming behavior, thus confirming the prosocial effect of MDMA in macaques. Additionally, at 1.5 and 2.0 mg/kg MDMA injection substantially decreases foraging behavior, which is consistent with the known anorexigenic effect of this compound. Furthermore, at 2.0 mg/kg MDMA injection induces an increase in locomotor behavior, which is also in accordance with its known stimulant property. Interestingly, MDMA injected at 1.0mg/kg increases the rate of object play, which might be interpreted as a decrease of the inhibition to manipulate a unique object in presence of others, or, as an increase of the intrinsic motivation to manipulate this object. Together, our results support the effectiveness of MDMA to study the complex neurobiology of primates’ social behaviors.     

SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor‐like proteins

Receptor‐like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine‐rich repeats (LRRs) and, in contrast with receptor‐like kinases (RLKs), lack a cytoplasmic kinase required for the initiation of downstream signalling. Recent studies have revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1‐1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf‐4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf‐4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked down by virus‐induced gene silencing, showed that the LRR domain as well as the kinase activity of SOBIR1 are required for the Cf‐4/Avr4‐triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitation of Cf‐4 accumulation. Together, these results suggest that, in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for the initiation of downstream signalling through its kinase domain.