In depth evaluation of the prognostic and predictive utility of PTEN immunohistochemistry in colorectal carcinomas: performance of three antibodies with emphasis on intracellular and intratumoral heterogeneity.

BACKGROUND: Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) loss of function is frequently detected in advanced colorectal cancer. Its detection is thought to have prognostic significance and it is being considered to predict responsiveness to anti-EGFR therapy. Unfortunately, while immunohistochemical assessment of PTEN expression is widespread, it lacks standardization and the results are hardly comparable across the available publications. METHODS: Retrospectively collected, formalin-fixed and paraffin-embedded colorectal tumor tissue samples from 55 patients were combined into tissue microarray (TMA) blocks. We used three different PTEN antibodies to determine the frequency, intensity and intracellular pattern of PTEN immunohistochemical labeling: Neomarkers, Dako and CellSignaling. We evaluated the aforementioned parameters in selected regions of colorectal cancers and in their lymph node metastases by using three scoring methods that take into consideration both staining frequency and intensity (H1-H3-score). We also evaluated intracellular localization. RESULTS: The Dako and CellSignaling antibodies stained predominantly cytoplasms, while the Neomarkers antibody specifically stained cell nuclei. PTEN H-scores were significantly lower in all tumor areas as compared to the normal colonic mucosa based on staining with the DAKO and CellSignaling antibodies. Intratumoral regional differences or differences between matching tumors and metastases were not detected with any of the antibodies. Neither Dako, neither CellSignaling, nor the Neomarkers antibodies revealed a significant correlation between PTEN expression and pT, Dukes/MAC and clinical stage. KRAS status, histological grade correlated with PTEN H-scores based on staining with the Neomarkers antibody. PTEN H-scores did not correlate with MMR status. PTEN H-scores did not show any correlation with relapse-free survival based on staining with either antibody. CONCLUSIONS: While PTEN expression decreased in colorectal cancer according to two antibodies, neither of the three applied PTEN antibodies could justify significant correlation with clinicopathological data, nor had prognostic value. Thus, we might conclude that immunohistochemical PTEN investigation remains a challenge requiring more standardized evaluation on larger number of cases to clarify its utility as a prognostic and predictive tool in CRC. The standardization of immunohistochemical method is key in the evaluation process, which is further discussed

Increased Mast Cell Density and Airway Responses to Allergic and Non-Allergic Stimuli in a Sheep Model of Chronic Asthma

BACKGROUND: Increased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: MC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MC(T) and MC(TC) respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase. MC(T) and MC(TC) density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MC(TC)/MC(T) ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MC(T) and MC(TC) density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MC(T) density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01). CONCLUSIONS: MC(T) and MC(TC) density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation

Bilateral effects of unilateral cerebellar lesions as detected by voxel based morphometry and diffusion imaging

Over the last decades, the importance of cerebellar processing for cortical functions has been acknowledged and consensus was reached on the strict functional and structural cortico-cerebellar interrelations. From an anatomical point of view strictly contralateral interconnections link the cerebellum to the cerebral cortex mainly through the middle and superior cerebellar peduncle. Diffusion MRI (dMRI) based tractography has already been applied to address cortico-cerebellar-cortical loops in healthy subjects and to detect diffusivity alteration patterns in patients with neurodegenerative pathologies of the cerebellum. In the present study we used dMRI-based tractography to determine the degree and pattern of pathological changes of cerebellar white matter microstructure in patients with focal cerebellar lesions. Diffusion imaging and high-resolution volumes were obtained in patients with left cerebellar lesions and in normal controls. Middle cerebellar peduncles and superior cerebellar peduncles were reconstructed by multi fiber diffusion tractography. From each tract, measures of microscopic damage were assessed, and despite the presence of unilateral lesions, bilateral diffusivity differences in white matter tracts were found comparing patients with normal controls. Consistently, bilateral alterations were also evidenced in specific brain regions linked to the cerebellum and involved in higher-level functions. This could be in line with the evidence that in the presence of unilateral cerebellar lesions, different cognitive functions can be affected and they are not strictly linked to the side of the cerebellar lesion

Healthcare-associated viral and bacterial infections in dentistry

Infection prevention in dentistry is an important topic that has gained more interest in recent years and guidelines for the prevention of cross-transmission are common practice in many countries. However, little is known about the real risks of cross-transmission, specifically in the dental healthcare setting. This paper evaluated the literature to determine the risk of cross-transmission and infection of viruses and bacteria that are of particular relevance in the dental practice environment. Facts from the literature on HSV, VZV, HIV, Hepatitis B, C and D viruses, Mycobacterium spp., Pseudomonas spp., Legionella spp. and multi-resistant bacteria are presented. There is evidence that Hepatitis B virus is a real threat for cross-infection in dentistry. Data for the transmission of, and infection with, other viruses or bacteria in dental practice are scarce. However, a number of cases are probably not acknowledged by patients, healthcare workers and authorities. Furthermore, cross-transmission in dentistry is under-reported in the literature. For the above reasons, the real risks of cross-transmission are likely to be higher. There is therefore a need for prospective longitudinal research in this area, to determine the real risks of cross-infection in dentistry. This will assist the adoption of effective hygiene procedures in dental practice

Actions of a Proline Analogue, L-Thiazolidine-4-Carboxylic Acid (T4C), on Trypanosoma cruzi

It is well established that L-proline has several roles in the biology of trypanosomatids. In Trypanosoma cruzi, the etiological agent of Chagas' disease, this amino acid is involved in energy metabolism, differentiation processes and resistance to osmotic stress. In this study, we analyzed the effects of interfering with L-proline metabolism on the viability and on other aspects of the T. cruzi life cycle using the proline analogue L- thiazolidine-4-carboxylic acid (T4C). The growth of epimastigotes was evaluated using different concentrations of T4C in standard culture conditions and at high temperature or acidic pH. We also evaluated possible interactions of this analogue with stress conditions such as those produced by nutrient starvation and oxidative stress. T4C showed a dose-response effect on epimastigote growth (IC50 = 0.89±0.02 mM at 28°C), and the inhibitory effect of this analogue was synergistic (p<0.05) with temperature (0.54±0.01 mM at 37°C). T4C significantly diminished parasite survival (p<0.05) in combination with nutrient starvation and oxidative stress conditions. Pre-incubation of the parasites with L-proline resulted in a protective effect against oxidative stress, but this was not seen in the presence of the drug. Finally, the trypomastigote bursting from infected mammalian cells was evaluated and found to be inhibited by up to 56% when cells were treated with non-toxic concentrations of T4C (between 1 and 10 mM). All these data together suggest that T4C could be an interesting therapeutic drug if combined with others that affect, for example, oxidative stress. The data also support the participation of proline metabolism in the resistance to oxidative stress

Response of cell wall composition and RNA-seq transcriptome to methyl-jasmonate in Brachypodium distachyon callus

Main conclusion: Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage. Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five–sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling

Phlebotominae (Diptera: Psycodidae) fauna in the Chaco region and Cutaneous Leishmaniasis transmission patterns in Argentina

In Argentina, the incidence of American Cutaneous Leishmaniasis (ACL) has shown a steady increase over the last few decades. In the Chaco biogeographical region, specifically, several outbreaks of ACL were recently reported in addition to the usual time-space scattering of ACL cases. However, little is known about the sandfly composition in the eastern, humid Chaco (HC) region or the western, dry Chaco (DC) region. Therefore, phlebotomine captures were performed throughout this region and an analysis of the distribution of reported ACL cases was conducted in order to assess the vector diversity in ACL endemic and epidemic scenarios in the Chaco region. The results support the hypothesis of two distinct patterns: (1) the DC, where Lutzomyia migonei was the most prevalent species, had isolated ACL cases and a zoonotic cycle; (2) the HC, where Lutzomyia neivai was the most prevalent species, had an increase in ACL incidence and outbreaks and an anthropozoonotic cycle. The epidemic risk in the Chaco region may be associated with the current climate trends, landscape modification, connection with other ACL foci, and Lu. neivai predominance and abundance. Therefore, changes in sandfly population diversity and density in the Chaco region are an indicator of emergent epidemic risk in sentinel capture sites

The mystery of the cerebellum: clues from experimental and clinical observations

Abstract The cerebellum has a striking homogeneous cytoarchitecture and participates in both motor and non-motor domains. Indeed, a wealth of evidence from neuroanatomical, electrophysiological, neuroimaging and clinical studies has substantially modified our traditional view on the cerebellum as a sole calibrator of sensorimotor functions. Despite the major advances of the last four decades of cerebellar research, outstanding questions remain regarding the mechanisms and functions of the cerebellar circuitry. We discuss major clues from both experimental and clinical studies, with a focus on rodent models in fear behaviour, on the role of the cerebellum in motor control, on cerebellar contributions to timing and our appraisal of the pathogenesis of cerebellar tremor. The cerebellum occupies a central position to optimize behaviour, motor control, timing procedures and to prevent body oscillations. More than ever, the cerebellum is now considered as a major actor on the scene of disorders affecting the CNS, extending from motor disorders to cognitive and affective disorders. However, the respective roles of the mossy fibres, the climbing fibres, cerebellar cortex and cerebellar nuclei remains unknown or partially known at best in most cases. Research is now moving towards a better definition of the roles of cerebellar modules and microzones. This will impact on the management of cerebellar disorders