Electron microscopic analysis in the gecko Lygodactylus reveals variations in micro-ornamentation and sensory organs distribution in the epidermis that indicate regional functions.

Possible pattern variations of micro-ornamentation in different areas of the skin in the gecko Lygodactylus have been analyzed by scanning and transmission electron microscopy. A map of micro-ornamentation present in various areas of the skin has been obtained. Differences in micro-ornamentation pattern and sensory organ distribution were detected. The "spinulated pattern" consists of shorter spinulae in dorsal versus ventral scales, and spinules are shorter in inner scale surface and hinge regions with respect to the outer scale surface. The spines derive from the accumulation of struts of corneous material mainly composed of corneous beta proteins (CBPs, formerly indicated as beta-keratins) that merge into pointed micro-ornamentation. The 3D-accumulation of CBPs within Oberhautchen cells can vary in some regions of different scales during Oberhautchen-beta cell differentiation, perhaps also under physical tensile forces derived from continuous scale growth. Three other main patterns of micro-ornamentation were detected and indicated as "corneous belts," "corneous dendritic ramification," and "serpentine-pit and groove." These variations from the typical spinulated pattern present in gecko epidermis are interpreted as transitional regions where the accumulation of corneous material in Oberhautchen cells that merges with underlying beta-cells gives rise to nonspinulated surfaces. Spinulated sensory organs with bristles and lenticular-shaped or knob-like tactile corpuscles are more numerous in ventral scales of the tail tip close to adhesive pads and near the digital pads. These regions are likely those most involved in the fine control of movements and response to vibrational stimuli derived from air and objects movements, including potential preys or predators.

Impact of Pre-Analytical Factors on MSI Test Accuracy in Mucinous Colorectal Adenocarcinoma: A Multi-Assay Concordance Study.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla™ and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla™ platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla™ platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off >50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla™ platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage >50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) ≥4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

Automated Workflow for Somatic and Germline Next Generation Sequencing Analysis in Routine Clinical Cancer Diagnostics.

Thanks to personalized medicine trends and collaborations between industry, clinical research groups and regulatory agencies, next generation sequencing (NGS) is turning into a common practice faster than one could have originally expected. When considering clinical applications of NGS in oncology, a rapid workflow for DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue samples, as well as producing high quality library preparation, can be real challenges. Here we consider these targets and how applying effective automation technology to NGS workflows may help improve yield, timing and quality-control. We firstly evaluated DNA recovery from archived FFPE blocks from three different manual extraction methods and two automated extraction workstations. The workflow was then implemented to somatic (lung/colon panel) and germline (BRCA1/2) library preparation for NGS analysis exploiting two automated workstations. All commercial kits gave good results in terms of DNA yield and quality. On the other hand, the automated workstation workflow has been proven to be a valid automatic extraction system to obtain high quality DNA suitable for NGS analysis (lung/colon Ampli-seq panel). Moreover, it can be efficiently integrated with an open liquid handling platform to provide high-quality libraries from germline DNA with more reproducibility and high coverage for targeted sequences in less time (BRCA1/2). The introduction of automation in routine workflow leads to an improvement of NGS standardization and increased scale up of sample preparations, reducing labor and timing, with optimization of reagents and management.

Effects of KEAP1 Silencing on the Regulation of NRF2 Activity in Neuroendocrine Lung Tumors.

BACKGROUND: The KEAP1/NRF2 pathway has been widely investigated in tumors since it was implicated in cancer cells survival and therapies resistance. In lung tumors the deregulation of this pathway is mainly related to point mutations of KEAP1 and NFE2L2 genes and KEAP1 promoter hypermethylation, but these two genes have been rarely investigated in low/intermediate grade neuroendocrine tumors of the lung. METHODS: The effects of KEAP1 silencing on NRF2 activity was investigated in H720 and H727 carcinoid cell lines and results were compared with those obtained by molecular profiling of KEAP1 and NFE2L2 in a collection of 47 lung carcinoids. The correlation between methylation and transcript levels was assessed by 5-aza-dC treatment. RESULTS: We demonstrated that in carcinoid cell lines, the KEAP1 silencing induces an upregulation of NRF2 and some of its targets and that there is a direct correlation between KEAP1 methylation and its mRNA levels. A KEAP1 hypermethylation and Loss of Heterozygosity at KEAP1 gene locus was also observed in nearly half of lung carcinoids. CONCLUSIONS: This is the first study that has described the effects of KEAP1 silencing on the regulation of NRF2 activity in lung carcinoids cells. The epigenetic deregulation of the KEAP1/NRF2 by a KEAP1 promoter hypermethylation system appears to be a frequent event in lung carcinoids.

Morphology of setae in regenerating caudal adhesive pads of the gecko Lygodactylus capensis (Smith, 1849).

After tail loss in the African gecko Lygodactylus capensis (Smith, 1949) a new tail is regenerated, including caudal adhesive pads. The axial skeleton of the regenerating tail consists in an elastic cartilaginous tube replacing the original vertebrae that allows interacting with the substrate like in the original tail. The formation of adhesive setae has been analyzed using transmission and scanning electron microscopy coupled to immunolabeling for Corneous Beta Proteins. During progressive stages of epidermal differentiation new setae are developed at stage 4 of the shedding cycle and contain Corneous Beta Proteins. These structural proteins are faintly localized in the Oberhäutchen but are abundant in the beta-layer, indicating that the two epidermal layers have a different protein composition. The setae originate from the growth of Oberhäutchen spinulae into the cytoplasm of clear cells and the latter produce a thick fibrous meshwork of keratin and other unknown proteins localized around the growing setae. This cytoskeleton likely allows molding tail setae like for digital setae. A graded development of setae is observed from the base to the tip of regenerated pads and from the periphery to more central areas. The terminal end of the setae is subdivided into numerous filamentous spatulae that increase the adhesion contact. Sensory boutons are frequently detected at the margin of tail scales and adhesive pads, likely improving compliance with the substrate. The present study indicates that tail regeneration is a convenient experimental model to analyze adhesive setae formation, microstructures that allow to these lizards climbing vertical and arboreal substrates.

Frequent NRG1 fusions in Caucasian pulmonary mucinous adenocarcinoma predicted by Phospho-ErbB3 expression.

NRG1 fusions were recently reported as a new molecular feature of Invasive Mucinous Adenocarcinoma (IMA) of the lung. The NRG1 chimeric ligand acts as a strong inductor of phosphorylation and tyrosine kinase activity of the ErbB2/ErbB3 heterodimer, thus enhancing the PI3K-AKT/MAPK pathways. The NRG1 fusions were widely investigated in Asian IMA cohorts, whereas just anecdotal information are available about the occurrence of NRG1 fusions in IMA Caucasian population. Here we firstly explored a large Caucasian cohort of 51 IMAs and 34 non-IMA cases for the occurrence of NRG1 rearrangements by fluorescent in situ hybridization (FISH) and RNA target sequencing. FISH results were correlated to the immunohistochemical expression of phosphorylated-ErbB3 (pErbB3) receptor and the mutational status of KRAS, EGFR and ALK genes. The NRG1 rearrangements were detected in 31% IMAs and 3% non-IMAs and the CD74-NRG1 fusion transcript variant was characterized in 4 NRG1-positive IMAs. Moreover, pErbB3 expression was found to be strictly associated to the mucinous pattern (p = 0.012, Chi-square test) and all IMA cases showing aberrant expression of pErbB3 demonstrated NRG1 rearrangements. No significant correlation between NRG1 rearrangements and EGFR, KRAS or ALK mutations respectively, was observed. We report for the first time that NRG1 fusions are driver alterations clearly associated with mucinous lung adenocarcinoma subtype of Caucasian patients and not exclusive of Asiatic population. pErbB3 immunostaining may represent a strong predictor of NRG1 fusions, pointing out the detection of pErbB3 by IHC as a rapid and effective pre-screening method to select the NRG1-positive patients.

New Indo-Pacific species of the genus Teretia Norman, 1888 (Gastropoda: Raphitomidae).

Four new species are assigned to the genus Teretia Norman, 1888 in the family Raphitomidae Bellardi, 1875 and herein described: Teretia neocaledonica sp. nov., T. sysoevi sp. nov., T. tongaensis sp. nov. from the southeastern Pacific and Teretia tavianii sp. nov. from the Gulf of Aden. The new species represent the first Indo-Pacific record of a genus previously known in the recent molluscan fauna by only two species from the Atlantic Ocean-Mediterranean Sea and Southern Africa. A possible Tethyan origin for the genus Teretia is suggested. 

Two new Horaiclavus (Horaiclavidae, Conoidea) species from the Indo-Pacific region.

The genus Horaiclavus includes eight Holocene Indo-Pacific species (Appeltans et al. 2012). Herein, we describe two new species that resemble members of this genus in some aspects of shell morphology, but otherwise show features that suggest that they differ from "typical" Horaiclavus species. Descriptions and measurements were based on shells oriented spire up with the aperture facing the viewer and made using a Leica MS 5 Stereomicroscope for incident light and a 10× ocular micrometer. Scanning electron microscope (SEM) micrographs were taken using a Hitachi S-2400. The classification adopted here follows Bouchet et al. (2011). The following abbreviations are used in the text: MZB = Museo di Zoologia dell'Università di Bologna; MNHN = Muséum National d'Histoire Naturelle, Paris, France; NHMUK = The Natural History Museum of United Kingdom [formerly British Museum (Natural History)], London, United Kingdom; ZMA = Naturalis Biodiversity Center, Leiden, the Netherlands; ZRC = Zoological Reference Collection, Raffles Museum of Biodiversity Research, Singapore; a = aperture length; b = shell width; l = length; a/l = ratio of aperture length to total shell length; b/l = ratio of shell breadth to total length; dd= dead collected specimen(s); stn. = station.

Four new African turriform gastropods (Mollusca: Conoidea).

Four new species, belonging to four distinct conoidean families, are described from east Africa and Mozambique Channel. Iredalea adenensis sp. nov. (Drilliidae Olsson, 1964), from Gulf of Aden, and Buchema shearmani sp. nov. (Horaiclavidae Bouchet et al., 2011), from off Mogadishu (Somalia), both trawled by local fishermen, represent the first record of their respective genera in eastern Africa. Crassispira somalica sp. nov. (Pseudomelatomidae Morrison, 1965), also collected offshore from Modagishu (Somalia), represents the first eastern Africa species bearing "typical" Crassispira features. Tropidoturris vizcondei sp. nov. (Borsoniidae Bellardi, 1875), from the Mozambique Channel, increases the knowledge of a genus considered endemic to southeastern Africa.

Three new bathyal raphitomine gastropods (Mollusca: Conoidea) from the Indo-Pacific region.

Three new species of Conoidea are described from Red Sea, Gulf of Aden and Philippines. Awheaturris lozoueti sp. nov., from Philippines, is the first representative in the recent Indo-Pacific molluscan fauna of a hitherto Miocene fossil genus. Taranis adenensis sp. nov., from Gulf of Aden, is the first species certainly referable to genus Taranis Jeffreys, 1870 reported in the Gulf of Aden and the smallest described member of this genus in the Indo-Pacific region. Mioawateria vivens sp. nov. represents the first member of the genus Mioawateria Vella, 1954 reported in the Red Sea. The status of Mioawateria is discussed and photographs of its type species, Awateria (Mioawateria) personata Powell, 1942, from the Pliocene of New Zealand, are presented for the first time.