Use of Dufour's gland secretion in nest defence and brood nutrition by hover wasps (Hymenoptera, Stenogastrinae)

Social wasps of the subfamily Stenogastrinae produce an abdominal secretion that is used in two distinct biological contexts. First, the secretion plays an important role in larval nutrition where it serves as a substrate in which food is placed by the adults for eventual consumption by the larvae. Second, in several species, females apply the same secretion to the substrate on which their nests are constructed, where it constitutes a sticky barrier that defends the immature brood from predation by ants. This paper describes for the first time ant guard construction behaviour of three species of stenogastrine wasps belonging to the genera Eustenogaster and Liostenogaster. The identification of compounds making up these secretions was also performed by gas chromatography-mass spectrometry. Ant guards and brood secretions were similar, with saturated and unsaturated long chain hydrocarbons and alcohols as major components. We further confirm that the glandular source of abdominal secretion is the Dufour's gland. This gland contains the same hydrocarbons, and in the same proportions as ant guards and brood secretion. We discuss the fundamental importance of Dufour's gland secretion in the social life of these wasps by comparing species with and without ant guards within the subfamily.     

Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.     

Fluorescent labeling of antibody fragments using split GFP

Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.     

KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment

Background

Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients.

Methodology/Principal Findings

In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass.

Conclusions

The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.     

Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/10⁴ cells in SA and 3.02 ( ± 1.86 [SD]) copy/10⁴ cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/10⁴ cells and 4.09 ( ± 4.26 [SD]) copy/10⁴ cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.     

Real-time monitoring of PCR amplification of proto-oncogene c-MYC using a Ta₂O₅ electrolyte-insulator-semiconductor sensor

We present a new approach for real-time monitoring of PCR amplification of a specific sequence from the human c-MYC proto-oncogene using a Ta(2)O(5) electrolyte-insulator-semiconductor (EIS) sensor. The response of the fabricated EIS sensor to cycle DNA amplification was evaluated and compared to standard SYBR-green fluorescence incorporation, showing it was possible to detect DNA concentration variations with 30 mV/μM sensitivity. The sensor's response was then optimized to follow in real-time the PCR amplification of c-MYC sequence from a genomic DNA sample attaining an amplification profile comparable to that of a standard real-time PCR. Owing to the small size, ease of fabrication and low-cost, the developed Ta(2)O(5) sensor may be incorporated onto a microfluidic device and then used for real-time PCR. Our approach may circumvent the practical and economical obstacles posed by current platforms that require an external fluorescence detector difficult to miniaturize and incorporate into a lab-on-chip system.     

Reconstructing the history of residence strategies in Indo-European-speaking societies: neo-, uxori-, and virilocality

Linguists and archaeologists have used reconstructions of early Indo-European residence strategies to constrain hypotheses about the homeland and trajectory of dispersal of Indo-European languages; however, these reconstructions are largely based on unsystematic and a historical use of the linguistic and ethnographic evidence, coupled with substantial bias in interpretation. Here I use cross-cultural data in a phylogenetic comparative framework to reconstruct the pattern of change in residence strategies in the history of societies speaking Indo-European languages. The analysis provides evidence in support of prevailing virilocality with alternative neolocality for Proto-Indo-European, and that this pattern may have extended back to Proto-Indo-Hittite. These findings bolster interpretations of the archaeological evidence that emphasize the "non-matricentric" structure of early Indo-European society; however, they also counter the notion that early Indo-European society was strongly "patricentric." I discuss implications of these findings in the context of the archaeological and genetic evidence on prehistoric social organization.