Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).     

New Insight into Benign Tumours of Major Salivary Glands by Proteomic Approach

Major salivary gland tumours are uncommon neoplasms of the head and neck. The increase of precise pre-operative diagnosis is crucial for their correct management and the identification of molecular markers would surely improve the required accuracy. In this study we performed a comparative proteomic analysis of fine needle aspiration fluids of the most frequent benign neoplasms of major salivary glands, namely pleomorphic adenoma and Warthin''s tumour, in order to draw their proteomic profiles and to point out their significant features. Thirty-five patients submitted to parotidectomy were included in the study, 22 were identified to have pleomorphic adenoma and 14 Warthin''s tumour. Fine needle aspiration samples were processed using a two-dimensional electrophoresis/mass spectrometry-based approach. A total of 26 differentially expressed proteins were identified. Ingenuity software was used to search the biological processes to which these proteins belong and to construct potential networks. Intriguingly, all Warthin''s tumour up-regulated proteins such as Ig gamma-1 chain C region, Ig kappa chain C region and Ig alpha-1 chain C region and S100A9 were correlated to immunological and inflammatory diseases, while pleomorphic adenomas such as annexin A1, annexin A4, macrophage-capping protein, apolipoprotein E and alpha crystalline B chain were associated with cell death, apoptosis and tumorigenesis, showing different features of two benign tumours. Overall, our results shed new light on the potential usefulness of a proteomic approach to study parotid tumours and in particular up regulated proteins are able to discriminate two types of benign parotid lesions.     

Periodicity and environmental drivers of apical and lateral growth in a Cerrado woody species

Key message

Apical and lateral growth are seasonal in a Cerrado species, and these events are related to each other and linked with climatic and environmental features.

Abstract

In the Cerrado, a tropical ecosystem with seasonal rainfall, we investigated the timing of leaf production and cambial activity, and checked whether these features are related to each other and with climatic and environmental factors. Between September 2011 and December 2012, sampling of main stem and vegetative phenological observations of Kielmeyera grandiflora (Wawra) Saddi (Calophyllaceae) were done monthly to assess seasonality in leaf production and cambial activity, and to compare these features with each other. To check the relationship of bud opening and the onset of cambial activity with climatic and environmental features, the average temperature and day length, and the precipitation sum in a time window ranging from 1 to 30 days before the occurrence of these events were recorded, and the coefficient of variation was calculated. Leaf production and cambial activity were seasonal. Bud opening occurred in September 2011 and August 2012, during the dry season. The onset of cambial activity occurred in October both in 2011 and 2012, 1–2 months after bud opening, at the beginning of the rainy season. The cambium was dormant in May, during the rainy season. Photoperiod and temperature showed low coefficients of variation in the time window before bud opening and onset of cambial activity, while rainfall presented a high coefficient of variation. Thus, both apical and lateral growth are seasonal events in Cerrado species, and are related to each other. A set of climatic and environmental features is related with seasonal growth, among which photoperiod and temperature may be important in the regulation of these events.

     

Identification of RELMgamma,a novel resistin-like molecule with a distinct expression pattern

We have identified RELMgamma, a novel member of the resistin-like molecule/found in inflammatory zone (RELM/FIZZ) family in mice and rats. Microarray and real-time RT-PCR experiments revealed a repression of RELMgamma mRNA in nasal respiratory epithelium of cigarette smoke-exposed versus untreated rats. The analysis of the physiological tissue-specific expression revealed highest expression in hematopoietic tissues, suggesting a cytokine-like role for RELMgamma. RELMgamma is most closely related to RELMalpha/FIZZ1. Despite the high similarity, the expression properties of the two genes are clearly distinct. While RELMgamma (approved symbol retnlg) is expressed in rat white adipose tissue, minute to no expression of RELMalpha was detected in that system. Thus, previous reports analyzing RELMalpha expression in rat adipose tissue might have been influenced by cross-hybridization with RELMgamma. Finally we could demonstrate that white adipose tissue of mice shows strong RELMalpha expression but only low levels of RELMgamma, indicating a species-specific gene regulation.     

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.     

Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.     

Association of Individual Non-Steroidal Anti-Inflammatory Drugs and Chronic Kidney Disease: A Population-Based Case Control Study

Background

Non-steroidal anti-inflammatory agents (NSAIDs) are known to be associated with renal damage. No clear evidence exists regarding differential risk of chronic kidney disease (CKD), specifically, across various NSAIDs.

Aim

The aim of this population-based case-control study was to evaluate the association between use of individual NSAIDs and risk of CKD in a general population of Southern Italy.

Methods

A nested case-control study was carried out using the general practice Arianna database, identifying incident CKD patients as cases and matched controls from 2006 to 2011. The date of first CKD diagnosis was defined as the index date (ID). Conditional logistic regressions were performed to estimate the risk of CKD associated with NSAIDs by class and individual drugs as compared to non-use during different time windows (within one year, six or three months prior to ID), with the latter being defined as current users. Among current users, the effect of cumulative exposure to these drugs was evaluated.

Results

Overall, 1,989 CKD cases and 7,906 matched controls were identified. A statistically significant increase in the risk of CKD was found for current users of oxicams (adjusted OR: 1.68; 95% CI: 1.15-2.44) and concerning individual compounds, for ketorolac (adj. OR: 2.54; 95% CI: 1.45-4.44), meloxicam (adj. OR: 1.98; 95% CI: 1.01-3.87) and piroxicam (adj. OR: 1.95; 95% CI: 1.19-3.21).

Conclusions

The risk of CKD varies across individual NSAIDs. Increased risk has been found for ketorolac, which may precipitate subclinical CKD through acute renal damage, and long-term exposure to oxicams, especially meloxicam and piroxicam.     

A proteomic approach to study parathyroid glands

Parathyroid tumours are heterogeneous and in some cases the diagnosis may be difficult using histological features. In this study we used a two-dimensional electrophoresis (2D)/mass spectrometry (MS)-based approach to examine the global changes of parathyroid adenoma tissues protein profile compared to the parathyroid normal tissues. Validation of protein expression was performed by immunoblotting using specific antibodies. Ingenuity software was used to identify the biological processes to which these proteins belong and to construct a potential network. A total of 30 proteins were found to be differentially expressed, of which 22 resulted in being over-expressed. Proteins identified by 2D/MS/MS proteomics were classified into functional categories and a major change (≥ 2-fold) in terms of expression was found in proteins involved in response to biotic stimuli, cell organization and signal transduction. After Ingenuity analysis, 14-3-3 ζ/δ appears to be a key protein in the network of parathyroid adenoma, where it is linked to other proteins such as annexin A2, B box and SPRY domain-containing protein (BSPRY), p53 and epidermal growth factor receptor (EGFR). Our results suggest that the proteomic approach was able to differentiate the protein profiles of normal parathyroid and parathyroid adenoma and identify a panel of proteins which are differentially expressed. The functional role of these proteins in the network of intracellular pathways is discussed.