Next-Generation Sequencing Workflow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM? Platform

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

Tract‐tracing study of the extrabulbar Olfactory projections in the brain of some teleosts

The extrabulbar olfactory projections (EBOP) is a collection of nerve fibers that originate from primary olfactory receptor neurons. These fibers penetrate into the brain, bypassing the olfactory bulbs (OBs). While the presence of an EBOP has been well established in teleosts, here we morphologically characterize the EBOP structure in four species each with a different morphological relationship of OB with the ventral telencephalic area. Tract‐tracing methods (carbocyanine DiI/DIA and biocytin) were used. FMRFamide immunoreactive nervus terminalis (NT) components were also visualized to define any neuroanatomical relationship between the NT and EBOP. Unilateral DiI/DiA application to the olfactory chamber stained the entire olfactory epithelium, olfactory nerve fibers, and ipsilateral olfactory bulb. Labeled primary olfactory fibers running ventromedially as extrabulbar primary olfactory projections reached various regions of the secondary prosencephalon. Only in Moenkhausia sanctaefilomenae (no olfactory peduncle) did lipophilic tracer‐labeled fibers reach the ipsilateral mesencephalon. The combination of tracing techniques and FMRFamide immunohistochemistry revealed a substantial overlap of the label along the olfactory pathways as well as in the anterior secondary prosencephalon. However, FMRFamide immunoreactivity was never colocalized in the same cellular or fiber component as visualized using tracer molecules. Our results showed a certain uniformity in the neuroanatomy and extension of EBOP in all four species, independent of the pedunculate feature of the OBs. The present study also provided additional evidence to support the view that EBOP and FMRFamide immunoreactive components of the NT are separate anatomical entities. Microsc. Res. Tech. 78:268–276, 2015. © 2015 Wiley Periodicals, Inc.     

High concentration of phosphorus is a distinctive feature of myelin. An X‐Ray elemental microanalysis study using freeze‐fracture scanning electron microscopy of rat sciatic nerve

We have used rat sciatic nerves submitted to freezing and freeze‐fracture to determine the elemental composition of small domains of the peripheral nerve studied at high resolution by scanning electron microscopy. We found that myelin of Schwann cells is unique in its high content in phosphorus (P) that was more than 10 times higher than P measured in any other cells. This high concentration in P makes myelin chemistry suitable of monitoring at the subcellular level using the herein described methodology. Microsc. Res. Tech. 78:537–539, 2015. © 2015 Wiley Periodicals, Inc.     

Assessment of daylight in rooms with different architectural features

Results are presented from a parametric study that assessed the amount of daylight in rooms with different architectural features: the orientation, window size and visible glazing transmittance, room depth, external obstruction angle and site. Annual lighting simulations were run in order to understand how the daylight availability within a space changes as a function of the architectural features. A sub-dataset of the full result database is examined in detail for north- and south-facing rooms in Turin, north-west Italy, with a visible glazing transmittance of 70%. Each feature is analysed for its influence on the daylighting conditions. A simple graphical tool is presented to promote an easier reading of the results. This was developed to provide a synthesis of information to the design team. It shows the influence of preliminary design solutions on the amount of indoor daylight. This allows a design team to assess indoor daylighting from the earliest design phases onwards and to determine which combinations of architectural features are able to provide high, acceptable or low daylight levels within a room.     

Hexagrid – hexagonal tube structures for tall buildings: patterns,modeling, and design

This paper provides a first insight on tube configurations based on the hexagonal shape (hexagrid) for tall buildings. The idea is to investigate the mechanical properties of hexagrid to assess their applicability in tall buildings and to compare their potential efficiency to the more popular diagrid systems. For the above purposes, a general homogenization approach has been established for dealing with any structural patterns, and a methodology for characterizing the structural patterns from the mechanical point of view has been developed and specified for hexagrids and diagrids. Then on the basis of a simple stiffness criterion, a design procedure has been proposed and applied to a tall building case study, and several structural solutions (both hexagrids and diagrids) have been designed and assessed by varying the major geometrical parameters of the patterns. Copyright © 2015 John Wiley & Sons, Ltd.     

Mn+Sb2O3 Thermite/Intermetallic Delay Compositions

The binary Mn+Sb₂O₃ pyrotechnic composition was investigated for mining detonator time delay applications. EKVI thermodynamic modelling predicted two maxima in the adiabatic reaction temperature. The local maximum, at a manganese fuel content of ca. 36 wt‐%, corresponds to a pure thermite‐type redox reaction: 3 Mn+Sb₂O₃→3 MnO+2Sb. The overall maximum in the adiabatic reaction temperature (ca. 1640 K), at the fuel‐rich composition of 49 wt‐% Mn, is consistent with the reaction 5 Mn+Sb₂O₃→3 MnO+2 MnSb, i.e. a combination of the standard thermite with an additional exothermic intermetallic reaction. XRD analysis of combustion residues confirmed the formation of MnSb and Mn₂Sb for fuel‐rich compositions. Burn rates were measured using delay elements assembled into commercial detonators. The d₅₀ particle sizes were 23.4 and 0.92 μm for the Mn fuel and Sb₂O₃ oxidant powders, respectively. The delay elements comprised rolled lead tubes with a length of 44 mm and an outer diameter of 6.4 mm. The rolling action compacted the pyrotechnic compositions to 74 ± 2 % theoretical maximum density. The burning rate increased linearly from 4.2 to 9.4 mm s⁻¹ over the composition range 25–50 wt‐% Mn.