Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34 + cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript andor exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicingprocessing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expressionsplicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34 + cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processespathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Protein expression differences between lung adenocarcinoma and squamous cell carcinoma with brain metastasis

AIM: We investigated tissue biomarkers in non-small cell lung cancer (NSCLC) to find indicators of brain metastasis and peritumoral brain edema.

PATIENTS AND METHODS: Fifty-two cases were studied out of which 26 had corresponding brain metastatic tissue. Clinicopathological characteristics of tumors were correlated with biomarkers of cell adhesion, cell growth, cell cycle and apoptosis regulation that were previously immunohistochemically studied but never analyzed separately according to histological subgroups, gender and smoking history.

RESULTS: Increased collagen XVII in adenocarcinoma (ADC) and increased caspase-9, CD44v6, and decreased cellular apoptosis susceptibility protein (CAS) and Ki-67 in squamous cell carcinoma (SCC) correlated significantly with brain metastasis. Increased β-catenin, E-cadherin and decreased caspase-9 expression in primary SCC, and decreased CD44v6 expression in brain metastatic SCC tissues showed a significant correlation with the extent of peritumoral brain edema. Positive correlation between smoking and biomarker expression could be observed in metastatic ADCs with p16 and caspase-8, while-negative correlation was found in SCC without brain metastasis with caspase-3, and in SCC with brain metastasis with p27.

CONCLUSION: Our results highlight the importance of separate analysis of biomarker expression in histological subtypes of NSCLC.

pLR-HL: a novel amphibian Bowman-Birk-type trypsin inhibitor from the skin secretion of the broad-folded frog, Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a "shotgun" cloning technique. It contains a disulphide loop between Cys5 and Cys15 which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesised by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nM. The substitution of Lys-8 by Phe (Phe8 -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μM. Additionally, both the disulphide loops of pLR-HL and Phe8 -pLR-HL were synthesised and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically-active moiety.

Molecular markers for predicting recurrence, progression and outcomes of bladder cancer (do the poster boys need new posters

Purpose of review
Molecular markers for bladder cancer recurrence and
progression continue to drive many research programmes.
Translating the laboratory findings into the clinical environment
where these markers are used in clinical decision making has
proved problematic. In the clinical arena, stage and grade are
still the main focus for decisions about patient management.
There is however an evolution in bladder cancer research from
single-marker/single-pathway research to a more global
assessment of the tumour cell with DNA microarrays and
proteomics.
Recent findings
In the last year, DNA microarray assessment has revealed
several interesting molecular markers such as p33ING1 and
DEK. Parallel ‘conventional’ single-pathway research has
focused on new novel markers such as HER2/neu, survivin and
matrix metalloproteinase 2 (MMP-2). Molecular markers that
have a long-standing association with bladder cancer
progression such as p53, E-cadherin and Ki-67 have been
reviewed by both single-marker studies and by microarray
studies and their status remains important.
Summary
It is an exciting time in the molecular biology research of bladder
cancer as the focus changes to assess the global genetic and
protein expression within tumour cells. From such a wealth of
information it is likely that molecular markers will make the
translation from benchside to bedside.

The Early Effects of Rapid Androgen Deprivation on Human Prostate Cancer

The androgen receptor (AR) is the dominant growth factor in prostate cancer (PCa). Therefore, understanding how ARs regulate the human transcriptome is of paramount importance. The early effects of castration on human PCa have not previously been studied 27 patients medically castrated with degarelix 7 d before radical prostatectomy. We used mass spectrometry, immunohistochemistry, and gene expression array (validated by reverse transcription-polymerase chain reaction) to compare resected tumour with matched, controlled, untreated PCa tissue. All patients had levels of serum androgen, with reduced levels of intraprostatic androgen at prostatectomy. We observed differential expression of known androgen-regulated genes (TMPRSS2, KLK3, CAMKK2, FKBP5). We identified 749 genes downregulated and 908 genes upregulated following castration. AR regulation of α-methylacyl-CoA racemase expression and three other genes (FAM129A, RAB27A, and KIAA0101) was confirmed. Upregulation of oestrogen receptor 1 (ESR1) expression was observed in malignant epithelia and was associated with differential expression of ESR1-regulated genes and correlated with proliferation (Ki-67 expression).

PATIENT SUMMARY: This first-in-man study defines the rapid gene expression changes taking place in prostate cancer (PCa) following castration. Expression levels of the genes that the androgen receptor regulates are predictive of treatment outcome. Upregulation of oestrogen receptor 1 is a mechanism by which PCa cells may survive despite castration.

A Statistical Characterization of Shadowed Fading in Indoor Off-Body Communications Channels At 5.8 GHz

This paper investigates the characteristics of the shadowed fading observed in off-body communications channels at 5.8 GHz using the κ-μ / gamma composite fading model. Realistic measurements have been conducted considering four individual scenarios namely line of sight (LOS) and non-LOS (NLOS) walking, rotation and random movements within an indoor laboratory environment. It is shown that the κ-μ / gamma composite fading model provides a better fit to the fading observed in off-body communications channels compared to the conventional Nakagami-m and Rician fading models.

Signal Reliability Improvement Using Selection Combining Based Macro-Diversity for Off-Body Communications At 868 MHz

This paper investigates the potential improvement in signal reliability for outdoor short-range off-body communications channels at 868 MHz using the macro-diversity offered by multiple co-located base stations. In this study, ten identical hypothetical base stations were positioned equidistantly around the perimeter of a rectangle of length 6.67 m and width 3.3 m. A body worn node was placed on the central chest region of an adult male. Five scenarios, each considering different user trajectories, were then analyzed to test the efficacy of using macro-diversity when the desired link is subject to shadowing caused by the human body. A number of selection combining based macro-diversity configurations consisting of four and then ten base stations were considered. It was found that using a macro-diversity system consisting of four base stations (or equivalently signal branches), a maximum diversity gain of 22.5 dB could be obtained while implementing a 10-base station setup this figure could be improved to 25.2 dB.

Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics

Immunohistochemistry (IHC) is a widely available and highly utilised tool in diagnostic histopathology and is used to guide treatment options as well as provide prognostic information. IHC is subjected to qualitative and subjective assessment, which has been criticised for a lack of stringency, while PCR-based molecular diagnostic validations by comparison are regarded as very rigorous. It is essential that IHC tests are validated through evidence-based procedures. With the move to ISO15189 (2012), not just of the accuracy, specificity and reproducibility of each test need to be determined and managed, but also the degree of uncertainty and the delivery of such tests. The recent update to ISO 15189 (2012) states that it is appropriate to consider the potential uncertainty of measurement of the value obtained in the laboratory and how that may impact on prognostic or predictive thresholds. In order to highlight the problems surrounding IHC validity, we reviewed the measurement of Ki67and p53 in the literature. Both of these biomarkers have been incorporated into clinical care by pathology laboratories worldwide. The variation seen appears excessive even when measuring centrally stained slides from the same cases. We therefore propose in this paper to establish the basis on which IHC laboratories can bring the same level of robust validation seen in the molecular pathology laboratories and the principles applied to all routine IHC tests.

Expression of proteins associated with hypoxia and Wnt pathway activation is of prognostic significance in hepatocellular carcinoma

In the development and progression of hepatocellular carcinoma, tumor hypoxia plays an important role, as does activation of the Wnt pathway. The aim of this study was to characterize the expression and interrelationship between hypoxia and Wnt-pathway-associated proteins as prognostic factors for hepatocellular carcinoma. Expression of HIF-1α, CA-IX, E-cadherin, β-catenin, and Ki-67 was assessed by immunohistochemistry in 179 primary hepatocellular carcinoma cases. Univariate and multivariate analyses were performed to assess the relationship between the clinicopathological factors, protein expression, overall survival (OS), and recurrence-free survival (RFS). By univariate analysis, tumor stage, size, satellitosis, and vascular invasion were confirmed as prognostic factors for worse OS and RFS. High expression of HIF-1α, CA-IX, β-catenin, Ki-67, and E-cadherin was observed in 60, 15, 64, 8, and 64 % of tumors, respectively, and this was significantly associated with poor OS. CA-IX, HIF-1α, and E-cadherin were independent predictors of poor prognosis. We stratified 169 patients into four groups according to the expression level of hypoxia and Wnt pathway markers. The group with high expression of both hypoxia and Wnt-pathway-associated proteins showed worst OS. The poor survival of this group was also significant in patients with early stage disease and tumor size of less than 5 cm (p < 0.05). We identified a subgroup of hepatocellular carcinoma patients with high expression of both hypoxia and Wnt pathway proteins and found this predictive of poor survival. The therapeutic options for this group might need to be revisited.

The κ− μ / Inverse Gamma Fading Model

Statistical distributions have been extensively used in modeling fading effects in conventional and modern wireless communications. In the present work, we propose a novel κ − µ composite shadowed fading model, which is based on the valid assumption that the mean signal power follows the inverse gamma distribution instead of the lognormal or commonly used gamma distributions. This distribution has a simple relationship with the gamma distribution, but most importantly, its semi heavy-tailed characteristics constitute it suitable for applications relating to modeling of shadowed fading. Furthermore, the derived probability density function of the κ − µ / inverse gamma composite distribution admits a rather simple algebraic representation that renders it convenient to handle both analytically and numerically. The validity and utility of this fading model are demonstrated by means of modeling the fading effects encountered in body centric communications channels, which have been known to be susceptible to the shadowing effect. To this end, extensive comparisons are provided between theoretical and respective real-time measurement results. It is shown that these comparisons exhibit accurate fitting of the new model for various measurement set ups that correspond to realistic communication scenarios.

The η − µ / Inverse Gamma Composite Fading Model

In this paper we propose a new composite fadingmodel which assumes that the mean signal power of an η−µ signalenvelope follows an inverse gamma distribution. The inversegamma distribution has a simple relationship with the gammadistribution and can be used to model shadowed fading due to itssemi heavy-tailed characteristics. To demonstrate the utility of thenew η−µ / inverse gamma composite fading model, we investigatethe characteristics of the shadowed fading behavior observed inbody centric communications channels which are known to besusceptible to shadowing effects, particularly generated by thehuman body. It is shown that the η−µ / inverse gamma compositefading model provided an excellent fit to the measurement data.Moreover, using Kullback-Leibler divergence, the η −µ / inversegamma composite fading model was found to provide a better fitto the measured data than the κ − µ / inverse gamma compositefading model, for the communication scenarios considered here.

Shadowed Fading in Indoor Off-Body Communications Channels: A Statistical Characterization using the κ – μ / Gamma Composite Fading Model

This paper investigates the characteristics of the shadowed fading observed in off-body communications channels at 5.8 GHz. This is realized with the aid of the $\kappa-\mu$ / gamma composite fading model which assumes that the transmitted signal undergoes $\kappa-\mu$ fading which is subject to \emph{multiplicative} shadowing. Based on this, the total power of the multipath components, including both the dominant and scattered components, is subject to non-negligible variations that follow the gamma distribution. For this model, we present an integral form of the probability density function (PDF) as well as important analytic expressions for the PDF, cumulative distribution function, moments and moment generating function. In the case of indoor off-body communications, the corresponding measurements were carried out in the context of four explicit individual scenarios namely: line of sight (LOS) and non-LOS (NLOS) walking, rotational and random movements. The measurements were repeated within three different indoor environments and considered three different hypothetical body worn node locations. With the aid of these results, the parameters for the $\kappa-\mu$ / gamma composite fading model were estimated and analyzed extensively. Interestingly, for the majority of the indoor environments and movement scenarios, the parameter estimates suggested that dominant signal components existed even when the direct signal path was obscured by the test subject's body. Additionally, it is shown that the $\kappa-\mu$ / gamma composite fading model provides an adequate fit to the fading effects involved in off-body communications channels. Using the Kullback-Leibler divergence, we have also compared our results with another recently proposed shadowed fading model, namely the $\kappa-\mu$ / lognormal LOS shadowed fading model. It was found that the $\kappa-\mu$ / gamma composite fading model provided a better fit for the majority of the scenarios considered in this study.

Switched Diversity Techniques for Indoor Off-body Communications Channels: An Experimental Analysis and Modeling

This paper investigates the potential improvement in signal reliability for indoor off-body communications channels operating at 5.8 GHz using switched diversity techniques. In particular we investigate the performance of switch-and-stay combining (SSC), switch-and-examine combining (SEC) and switch-and-examine combining with post-examining selection (SECps) schemes which utilize multiple spatially separated antennas at the base station. During the measurements a test subject, wearing an antenna on his chest, performed a number of walking movements towards and then away from a uniform linear array. It was found that all of the considered diversity schemes provided a worthwhile signal improvement. However, the performance of the diversity systems varied according to the switching threshold that was adopted. To model the fading envelope observed at the output of each of the combiners, we have applied diversity specific equations developed under the assumption of Nakagami-$m$ fading. As a measure of the goodness-of-fit, the Kullback-Leibler divergence between the empirical and theoretical probability density functions (PDFs) was calculated and found to be close to 0. To assist with the interpretation of the goodness-of-fit achieved in this study, the standard deviation, $\sigma$, of a zero-mean, $\sigma^2$ variance Gaussian PDF used to approximate a zero-mean, unit variance Gaussian PDF is also presented. These were generally quite close to 1 indicating that the theoretical models provided an adequate fit to the measured data.

Role on serine protease inhibitors in the pathogenisis of Steinernema carpocapsae

Tese de Doutoramento, Biologia (Biologia Celular e Molecular), 18 de Novembro de 2013, Universidade dos Açores.

Propagação de fendas por fadiga quando sujeitas a carregamentos em Modo I ou Modo III

A propagação de fenda sob fadiga pode ser observada em vários componentes mecânicos, que podem ser feitos de chapa fina ou espessa (ou cascas) e, consequentemente, serem sujeitos a estado plano de tensão ou estado plano de deformação, respectivamente. As cargas aplicadas num corpo sólido que contém um entalhe estreito ou uma fenda aguda irão induzir uma zona de escoamento limite de material com uma dimensão que dependerá das propriedades mecânicas do material, bem como a espessura do corpo, o comprimento de fenda e a intensidade das cargas aplicadas. A propagação da fenda pode então ocorrer sob modo I, II, III ou modo misto. Esta tese apresenta as funções integrais JI, JII e JIII , que foram correlacionadas com os factores de intensidade de tensão KI, KII e KIII, para provetes CT espessos e finos. A avaliação dos valores do Integral-J foi feita para diferentes comprimentos de fenda, ao longo da frente de fenda, usando o Método dos Elementos Finitos (MEF), com nós colapsados e intermédios deslocados a ¼ do comprimento da aresta, com o objectivo de simular a singularidade na extremidade da fenda. A interacção entre os modos de abertura, corte e rasgamento é também discutida. Adicionalmente, a propagação de fenda sob o modo I e o modo III foi determinada experimentalmente, à temperatura ambiente, para um aço inoxidável austenítico Cr-Mn de alta resistência.