The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1‐C CRISPR2 is present in few strains but type 2‐A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre‐exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer‐associated motif‐shuffling demonstrated that the GG motif is sufficient to discriminate self and non‐self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II‐A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.     

Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry

A. Stacchini, P. Carucci, D. Pacchioni, G. Accinelli, A. Demurtas, S. Aliberti, M. Bosco, M. Bruno, A. Balbo Mussetto, M. Rizzetto, G. Bussolati and C. De Angelis
Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry Objective: Although endoscopic ultrasound combined with fine needle aspiration (EUS‐FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS‐guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. Methods: Of 1560 patients having EUS‐guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS‐FNA. There was adequate material to perform FC analysis for all but one case. Results: EUS‐FNA‐FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. Conclusions: Our results show that a combination of EUS‐FNA‐FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep‐seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non‐lymphoma cases.     

Efficient nitrogen-fixing <Emphasis Type="Italic">Rhizobium</Emphasis> strains isolated from amazonian soils are highly tolerant to acidity and aluminium

One of the most cultivated and consumed vegetables in Brazil is the common bean, Phaseolus vulgaris L. The symbiosis of this plant species with nitrogen-fixing bacteria that are adapted to the stresses commonly found in tropical soils can increase production. The aim of this study was to evaluate the symbiotic effectiveness of bacterial strains from soils under different land uses in the Amazon region. Further, rhizobia tolerance to acidity and aluminium and the involvement of some possible physiological mechanisms of such tolerance were also investigated. In assessing the efficiency of biological nitrogen fixation, inoculation with strains UFLA04-195, UFLA04-173 and UFLA04-202, belonging to the genus Rhizobium, resulted in greater plant growth, higher shoot nitrogen content and good nodulation compared to the inoculation with the strain CIAT 899 (R. tropici), and to the mineral nitrogen control or Burkholderia fungorum strains that nodulated or not bean plants. These efficient strains grew better at pH 5.0 than at pH 6.0 or pH 6.9; they also tolerated up to 1 mmol l⁻¹ of Al³⁺ and showed an increased production of exopolysaccharides where the growing rates were less (pH 6.0 and pH 6.9). With respect to aluminium, the highest production of EPS produced greater tolerance to this element. Taken together, these results indicate that the strains evaluated in this study were tolerant to acidity and aluminium; they appeared to have developed resistance mechanisms such as EPS production and a resistant cell outer membrane (indicated by resistance to polymyxin and methyl violet). As these strains also gave increased yields of the host species, further studies on whether to recommend these strains as inoculants are already underway.     

Implications of maternal nutrient restriction in transgenerational programming of hypertension and endothelial dysfunction across F1-F3 offspring

AimsAn extensive variety of prenatal insults are associated with an increased incidence of metabolic and cardiovascular disorders in adult life. We previously demonstrated that maternal global nutrient restriction during pregnancy leads to increased blood pressure and endothelial dysfunction in the adult offspring. This study aimed to assess whether prenatal exposure to nutritional insult has transgenerational effects in F₂ and F₃ offspring.Main methodsFor this, female Wistar rats were randomly divided into two groups on day 1 of pregnancy: a control group fed standard chow ad libitum and a restricted group fed 50% of the ad libitum intake throughout gestation. At delivery, all animals were fed a standard laboratory chow diet. At 11 weeks of age, one female and one male from each restricted litter were randomly selected and mated with rats from another restricted litters in order to generate the F₂ offspring. The same procedure produced F₃ generation. Similarly, the rats in the control group were bred for each generation.Key FindingsOur findings show that the deleterious effects of maternal nutrient restriction to which the F₀ mothers were exposed may not be limited to the male first generation. In fact, we found that elevated blood pressure, an impaired vasodilatory response to acetylcholine and alterations in NO production were all transferred to the subsequent males from F₂ and F₃ generations.SignificanceOur data show that global nutrient restriction during pregnancy results in a specific phenotype that can be passed transgenerationally to a second and third generation.     

A uniquely modern human pattern of endocranial development. Insights from a new cranial reconstruction of the Neandertal newborn from Mezmaiskaya

The globular braincase of modern humans is distinct from all fossil human species, including our closest extinct relatives, the Neandertals. Such adult shape differences must ultimately be rooted in different developmental patterns, but it is unclear at which point during ontogeny these group characteristics emerge.Here we compared internal shape changes of the braincase from birth to adulthood in Neandertals (N = 10), modern humans (N = 62), and chimpanzees (N = 62). Incomplete fossil specimens, including the two Neandertal newborns from Le Moustier 2 and Mezmaiskaya, were reconstructed using reference-based estimation methods. We used 3D geometric morphometrics to statistically compare shapes of virtual endocasts extracted from computed-tomographic scans. Throughout the analysis, we kept track of possible uncertainties due to the missing data values and small fossil sample sizes.We find that some aspects of endocranial development are shared by the three species. However, in the first year of life, modern humans depart from this presumably ancestral pattern of development. Newborn Neandertals and newborn modern humans have elongated braincases, and similar endocranial volumes. During a ‘globularization-phase’ modern human endocasts change to the globular shape that is characteristic for Homo sapiens. This phase of early development is unique to modern humans, and absent from chimpanzees and Neandertals.Our results support the notion that Neandertals and modern humans reach comparable adult brain sizes via different developmental pathways. The differences between these two human groups are most prominent directly after birth, a critical phase for cognitive development.     

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.     

Transgenesis applied to goat: current applications and ongoing research

Compared to experiments involving pigs, cows and/or sheep, transgenesis applied to goats is probably less advertised. However, recent successes and increasing amount of dedicated research make this species of special interest for ongoing biological and physiological questions on genome engineering in large animals. This short review aims at highlighting the current applications and limitations of the goat genome manipulation.     

Activation of c-Jun N-terminal kinase (JNK) during mitosis in retinal progenitor cells

Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.