Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by characterizing the disulfide containing intermediates using different experimental conditions and analytical techniques. So far, some aspects still remain unclear such as the role of the loop 65-72 in the folding pathway. We have studied the oxidative folding of a RNase A derivative containing at position 67 the substitution Asn --> isoAsp where the local structure of the loop 65-72 has been modified keeping intact the C65-C72 disulfide bond. By comparing the folding behavior of this mutant to that of the wild-type protein, we found that the deamidation significantly decreases the folding rate and alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.     

A new arthropod from the Silurian Konservat-Lagerstätte of Herefordshire, UK

A small, non-biomineralized, macrophagous arthropod with chelicerate affinities, Offacolus kingi gen. et sp. nov., from the Silurian (Wenlock Series) of Herefordshire, UK, is described. The dorsal exoskeleton comprises an arch-like cephalic shield, a thorax of three free tergites and a triangular posterior tagma of five fused tergites, the last with a stout postero-dorsally directed medial spine. Seven pairs of appendages beneath the cephalic shield surround a postero-medially sited oral cavity on the ventral surface of the head. Appendages I and, probably II are uniramous and project antero-ventrally; I was sensory and II sensory and/or ambulatory. Appendages III-VI are biramous, each with an antero-ventrally projecting ramus and a robust, highly geniculate, horizontally oriented ramus that projects through an anterior gape. The former rami were ambulatory and the latter have spinose terminal podomeres and functioned as a unit for trapping food and transferring it towards the oral cavity. Appendage VII, which is probably uniramous, is posteroventrally directed and flap like. Each tergite of the thorax and posterior tagma covers at least a pair (probably two pairs) of probably biramous appendages with each ramus flap like and setose.     

Mouse Mesenchyme forkhead 2 (Mf2): expression, DNA binding and induction by sonic hedgehog during somitogenesis

Cloning and sequencing of mouse Mf2 (mesoderm/mesenchyme forkhead 2) cDNAs revealed an open reading frame encoding a putative protein of 492 amino acids which, after in vitro translation, binds to a DNA consensus sequence. Mf2 is expressed at high levels in the ventral region of newly formed somites, in sclerotomal derivatives, in lateral plate and cephalic mesoderm and in the first and second branchial arches. Other regions of mesodermal expression include the developing tongue, meninges, nose, whiskers, kidney, genital tubercule and limb joints. In the nervous system Mf2 is transcribed in restricted regions of the mid- and forebrain. In several tissues, including the early somite, Mf2 is expressed in cell populations adjacent to regions expressing sonic hedgehog (Shh) and in explant cultures of presomitic mesoderm Mf2 is induced by Shh secreted by COS cells. These results suggest that Mf2, like other murine forkhead genes, has multiple roles in embryogenesis, possibly mediating the response of cells to signaling molecules such as SHH.     

Organization of the Human Frontal Pole Revealed by Large-Scale DTI-Based Connectivity: Implications for Control of Behavior

The goal of the current study was to examine the pattern of anatomical connectivity of the human frontal pole so as to inform theories of function of the frontal pole, perhaps one of the least understood region of the human brain. Rather than simply parcellating the frontal pole into subregions, we focused on examining the brain regions to which the frontal pole is anatomically and functionally connected. While the current findings provided support for previous work suggesting the frontal pole is connected to higher-order sensory association cortex, we found novel evidence suggesting that the frontal pole in humans is connected to posterior visual cortex. Furthermore, we propose a functional framework that incorporates these anatomical connections with existing cognitive theories of the functional organization of the frontal pole. In addition to a previously discussed medial-lateral distinction, we propose a dorsal-ventral gradient based on the information the frontal pole uses to guide behavior. We propose that dorsal regions are connected to other prefrontal regions that process goals and action plans, medial regions are connected to other brain regions that monitor action outcomes and motivate behaviors, and ventral regions connect to regions that process information about stimuli, values, and emotion. By incorporating information across these different levels of information, the frontal pole can effectively guide goal-directed behavior.     

Endopeptidase-Mediated Beta Lactam Tolerance

In many bacteria, inhibition of cell wall synthesis leads to cell death and lysis. The pathways and enzymes that mediate cell lysis after exposure to cell wall-acting antibiotics (e.g. beta lactams) are incompletely understood, but the activities of enzymes that degrade the cell wall (‘autolysins’) are thought to be critical. Here, we report that Vibrio cholerae, the cholera pathogen, is tolerant to antibiotics targeting cell wall synthesis. In response to a wide variety of cell wall- acting antibiotics, this pathogen loses its rod shape, indicative of cell wall degradation, and becomes spherical. Genetic analyses revealed that paradoxically, V. cholerae survival via sphere formation required the activity of D,D endopeptidases, enzymes that cleave the cell wall. Other autolysins proved dispensable for this process. Our findings suggest the enzymes that mediate cell wall degradation are critical for determining bacterial cell fate - sphere formation vs. lysis – after treatment with antibiotics that target cell wall synthesis.     

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Background

Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and Methods

PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results

PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions

Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.