Microreactors for peptide synthesis: looking through the eyes of twenty first century !!!

The twenty first century has witnessed several advances in synthetic chemistry, among them microreactors. It is expected that these devices will have a considerable impact on synthetic organic chemistry since they offer a wide range of applications in various fields. Perhaps the synthesis of peptides deserves mention in this regard as these molecules are emerging as therapeutics and offer several advantages over the so-called small molecules. This minireview does not aim to address microreactors in detail, but explains various peptide synthesis methods that involve microfluidic techniques, highlighting the need for further improvement and expansion of microdevices/microreactors.     

Prominent intraspecific genetic divergence within Anopheles gambiae sibling species triggered by habitat discontinuities across a riverine landscape

The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia river in West Africa. We characterized putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically associated population subdivisions within both species. Microsatellite loci on chromosome‐3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene flow was observed between An. gambiae populations west and east of an extensively rice‐cultivated region apparently colonized exclusively by An. coluzzii. Notably, this ‘intraspecific’ differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome‐X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human‐made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro‐tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.     

An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

Background

Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.

Methodology/Principal Findings

We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.

Conclusion

Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.     

Estimating Species Richness and Modelling Habitat Preferences of Tropical Forest Mammals from Camera Trap Data

Medium-to-large mammals within tropical forests represent a rich and functionally diversified component of this biome; however, they continue to be threatened by hunting and habitat loss. Assessing these communities implies studying species’ richness and composition, and determining a state variable of species abundance in order to infer changes in species distribution and habitat associations. The Tropical Ecology, Assessment and Monitoring (TEAM) network fills a chronic gap in standardized data collection by implementing a systematic monitoring framework of biodiversity, including mammal communities, across several sites. In this study, we used TEAM camera trap data collected in the Udzungwa Mountains of Tanzania, an area of exceptional importance for mammal diversity, to propose an example of a baseline assessment of species’ occupancy. We used 60 camera trap locations and cumulated 1,818 camera days in 2009. Sampling yielded 10,647 images of 26 species of mammals. We estimated that a minimum of 32 species are in fact present, matching available knowledge from other sources. Estimated species richness at camera sites did not vary with a suite of habitat covariates derived from remote sensing, however the detection probability varied with functional guilds, with herbivores being more detectable than other guilds. Species-specific occupancy modelling revealed novel ecological knowledge for the 11 most detected species, highlighting patterns such as ‘montane forest dwellers’, e.g. the endemic Sanje mangabey (Cercocebus sanjei), and ‘lowland forest dwellers’, e.g. suni antelope (Neotragus moschatus). Our results show that the analysis of camera trap data with account for imperfect detection can provide a solid ecological assessment of mammal communities that can be systematically replicated across sites.     

Boosting Long-Term Memory via Wakeful Rest: Intentional Rehearsal Is Not Necessary,Consolidation Is Sufficient

People perform better on tests of delayed free recall if learning is followed immediately by a short wakeful rest than by a short period of sensory stimulation. Animal and human work suggests that wakeful resting provides optimal conditions for the consolidation of recently acquired memories. However, an alternative account cannot be ruled out, namely that wakeful resting provides optimal conditions for intentional rehearsal of recently acquired memories, thus driving superior memory. Here we utilised non-recallable words to examine whether wakeful rest boosts long-term memory, even when new memories could not be rehearsed intentionally during the wakeful rest delay. The probing of non-recallable words requires a recognition paradigm. Therefore, we first established, via Experiment 1, that the rest-induced boost in memory observed via free recall can be replicated in a recognition paradigm, using concrete nouns. In Experiment 2, participants heard 30 non-recallable non-words, presented as ‘foreign names in a bridge club abroad’ and then either rested wakefully or played a visual spot-the-difference game for 10 minutes. Retention was probed via recognition at two time points, 15 minutes and 7 days after presentation. As in Experiment 1, wakeful rest boosted recognition significantly, and this boost was maintained for at least 7 days. Our results indicate that the enhancement of memory via wakeful rest is not dependent upon intentional rehearsal of learned material during the rest period. We thus conclude that consolidation is sufficient for this rest-induced memory boost to emerge. We propose that wakeful resting allows for superior memory consolidation, resulting in stronger and/or more veridical representations of experienced events which can be detected via tests of free recall and recognition.     

Permanent Cardiac Sarcomere Changes in a Rabbit Model of Intrauterine Growth Restriction

Background

Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood.

Methods and Results

IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts.

Conclusion

The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.     

Biochemical and MALDI analysis of the human sperm antigen gp20, homologue of leukocyte CD52.

In previous work we demonstrated that gp20, a sialoglycoprotein of human sperm is homologous to the leukocyte antigen CD52 and that anti-gp20 recognizes an antigen of the same molecular weight as that recognized by CAMPATH-1 (anti CD52) in leukocytes and sperm, but with some differences. In this study we used anti-gp20 to perform immunoblot analysis of many different sperm, seminal plasma and leukocyte samples. The sperm and seminal plasma antigens were similar and appeared to consist of two components, whereas the leukocyte antigen is unique. Evidence of the presence of two components of the sperm antigen, running respectively at about 19 and 21 kDa, was obtained by analyzing the purified antigen stained with Coomassie brilliant blue and by immunoblot analysis of the antigen after two-dimensional electrophoresis. Both components had an isoelectric point (pI) between 3 and 6. MALDI analysis of the purified antigen confirmed the presence of two components and indicated masses (Mr) of 8243 and 10908. The possible relationship between these findings and the presence of two forms of the CD52 gene differing at two aminoacids C-terminal to the GPI-anchor site has been discussed.     

Molecular validation of the acute phencyclidine rat model for schizophrenia: identification of translational changes in energy metabolism and neurotransmission

Administration of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist phencyclidine (PCP) to rodents is widely used as preclinical model for schizophrenia. Most studies on this model employ methods investigating behavior and brain abnormalities. However, little is known about the corresponding peripheral effects. In this study, we analyzed changes in brain and serum molecular profiles, together with alterations in behavior after acute PCP treatment of rats. Furthermore, abnormalities in peripheral protein expression of first and recent onset antipsychotic free schizophrenia patients were assessed for comparison with the preclinical model. PCP treatment induced hyperlocomotion and stereotypic behavior, which have been related to positive symptoms of schizophrenia. Multiplex immunoassay profiling of serum revealed molecular abnormalities similar to those seen in first and recent onset, antipsychotic free schizophrenia patients. Also, increased insulin levels were detected after administration of a glucose tolerance test (GTT), consistent with previous studies showing changes in insulin signaling in patients with schizophrenia. Finally, schizophrenia-relevant alterations in brain molecules were found in the hippocampus and to a lesser extent in the frontal cortex using liquid-chromatography mass spectrometry and (1)H nuclear magnetic resonance spectroscopy. In conclusion, this study identified behavioral and molecular alterations in the acute PCP rat model, which are also observed in human schizophrenia. We propose that the corresponding changes in serum in both animals and patients may have utility as surrogate markers in this model to facilitate discovery and development of novel drugs for treatment of certain pathological features of schizophrenia.