Description of immature stages of Thanatophilus sinuatus (Coleoptera: Silphidae).

Necrophagous beetles of genus Thanatophilus are well recognized as a group of beetles with a high potential utility in forensic entomology. They can be used to estimate postmortem interval (PMI) or validate the value for other groups of insects commonly encountered on human remains, like blowflies (Calliphoridae). However, reliable tools for instar and species identification of their larvae are needed as such information is crucial for allowing accurate PMI estimate. One of the most common species of the genus Thanatophilus in Europe is Thanatophilus sinuatus. This species occurs frequently on human remains and its larvae feed on decaying tissues throughout their development. Therefore, the larvae could become useful bioindicators for forensic entomology, although their current description does not allow reliable instar or species identification. Our goal was to provide morphological characters for species and instar identification of all larval stages of T. sinuatus. The larvae were obtained from laboratory rearing under controlled conditions (20 °C and 16:8 h of light/dark period). Qualitative and quantitative morphological instar and species-specific characters are described and illustrated. Additionally, we report observations of biological and developmental lengths for all stages of the species. We also compared these morphological characters with recent description of T. rugosus and provided an identification key of these two similar and often co-occurring species. We noticed that some characters for instar identification were shared between T. sinuatus and T. rugosus and were confirmed by comparison with larvae of T. dentigerus that they can be applied to other species of the genus.

Revisited larval morphology of Thanatophilus rugosus (Coleoptera: Silphidae).

Determination of insect species and their instars, occurring on human remains, is important information that allows us to use insects for estimation of postmortem interval and detect possible manipulation with the body. However, larvae of many common species can be identified only by molecular methods, which is not always possible. The instar determination is even more challenging, and qualitative characters that would allow a more precise identification are mostly unknown. Thanatophilus rugosus (Linnaeus, 1758) is a common necrophagous beetle in the whole Palaearctic region from Europe to Japan. The species is often encountered on corpses of large vertebrates including humans, and its potential to become a useful bioindicator for forensic entomology is therefore high. Adults can be easily distinguished from other species; however, larvae were never thoroughly described to allow species and instar identification. The aim of this study was to provide reliable morphological characters that would allow species and instar identification of T. rugosus larvae. The material for morphological study was obtained from rearing under controlled conditions (20 °C and 12:12 h of light/dark period), and specimens that were not studied morphologically were allowed to complete their development. Quantitative and qualitative morphological characters for instar and species identification are described and illustrated. Additionally, we report observations of biology and developmental length for all stages of the species.

Sequence-Specific Recognition of MicroRNAs and Other Short Nucleic Acids with Solid-State Nanopores.

The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of nontarget oligonucleotides.

Microbial degradation of N-methyl-2-pyrrolidone in surface water and bacteria responsible for the process.

Due to widespread utilization in many industrial spheres and agrochemicals, N-methyl-2-pyrrolidone (NMP) is a potential contaminant of different surface water ecosystems. Hence, investigation was made into its aerobic microbial degradability in samples of water from a river, wetland area and spring. The results showed that the compound was degradable in all water types, and that the fastest NMP removal occurred in 4 days in river water, while in the wetland and spring samples the process was relatively slow, requiring several months to complete. Key bacterial degraders were successfully isolated in all cases, and their identification proved that pseudomonads played a major role in NMP degradation in river water, while the genera Rhodococcus and Patulibacter fulfilled a similar task in the wetland sample. Regarding spring water, degrading members of the Mesorhizobium and Rhizobium genera were found.

Nanopore Analysis of Single-Stranded Binding Protein Interactions with DNA.

We study the binding of E. coli single-stranded binding protein (SSB) to single-stranded DNA (ssDNA) using a solid-state nanopore assay. We find that saturated nucleoprotein complexes can be distinguished easily from free SSB, ssDNA, or double-stranded DNA individually and demonstrate that the high affinity of SSB for ssDNA can be exploited to achieve high-fidelity differentiation from duplex molecules in a mixture. We then study nucleoprotein filament formation by systematically varying the amount of SSB relative to ssDNA. We observe a concomitant shift in the mean amplitude of electrical events that is consistent with weakly cooperative binding. Finally, we compare circular and linearized ssDNA saturated with SSB and use the results to infer structural details of the nucleoprotein complex.

Selective detection and quantification of modified DNA with solid-state nanopores.

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

N-methyl-2-pyrrolidone-degrading bacteria from activated sludge.

N-methyl-2-pyrrolidone (NMP) is a widely used solvent for many organic compounds and a component found in a vast array of chemical preparations. For this research paper, NMP degrading bacteria were isolated from two samples of activated sludge. They pertained to both Gram-negative and Gram-positive members, and belong to the Pseudomonas, Paracoccus, Acinetobacter and Rhodococcus genera. All the strains utilized 300 mg/L of NMP as the only source of carbon, energy and nitrogen over several days, and they were shown to additionally be able to degrade N-acetylphenylalanine (NAP). The growth of all the isolated strains was recorded at different NMP concentrations, to a maximum of 20 g/L.

Small-scale regional engineering geological study of the Czech Republic evaluating the relationship between slope gradients and engineering geological zones.

The aim of the small-scale regional engineering geological study of the Czech Republic was to evaluate the relationship between slope gradient and engineering geological zones. The research motivation was to determine the average slope gradient, 25%, 50% (median) and 75% quantiles related to the different engineering geological zones. This scientific information is critical from the perspectives of engineering geology, geotechnical engineering, and foundation engineering because an increasing slope gradient evokes the need to create a cut respectively foundation excavation or another excavation in the geological structure, which increases the probability of occurrence of the problem in terms of differential settlement and bearing capacity of the structures. The research was carried out in the territory of the Czech Republic in 8 Quaternary zones with soil foundation ground and 10 pre-Quaternary zones with rocks and semi-rocks and their eluvia. A significant difference in the statistical characteristics of slope gradients was found in the group of Quaternary engineering geological zones (evaluated group I) compared to the group of pre-Quaternary zones (evaluated group II). The value range of the average slope gradient was 1.65° (16.9%) to 5.89° (60.3%) for the Quaternary engineering geological zones (soil foundation ground), representing 43.4% difference. Whereas for the over-quaternary engineering geological zones (rocks, semi-rocks and their eluvia), the difference was much higher, 3.59° (36.8%) to 9.76° (100%-value determined as a referential because it was the maximum), which is also reflected in a more significant percentage difference of 63.2%.

Chemically synthesised atomically precise gold clusters deposited and activated on titania. Part II.

Synchrotron XPS was used to investigate a series of chemically synthesised, atomically precise gold clusters Au(n)(PPh3)y (n = 8, 9 and 101, y depending on the cluster size) immobilized on anatase (titania) nanoparticles. Effects of post-deposition treatments were investigated by comparison of untreated samples with analogues that have been heat treated at 200 °C in O2, or in O2 followed by H2 atmosphere. XPS data shows that the phosphine ligands are oxidised upon heat treatment in O2. From the position of the Au 4f(7/2) peak it can be concluded that the clusters partially agglomerate immediately upon deposition. Heating in oxygen, and subsequently in hydrogen, leads to further agglomeration of the gold clusters. It is found that the pre-treatment plays a crucial role in the removal of ligands and agglomeration of the clusters.

Integrative taxonomy and species distribution models of the genus Diamesus Hope, 1840 (Coleoptera: Staphylinidae: Silphinae).

Integrative taxonomy of Diamesus Hope, 1840 (Coleoptera: Silphinae) is presented. Adults of D. bimaculatus Portevin, 1914 (endemic to Taiwan) and D. osculans (Vigors, 1825) (widely distributed from northern India to Australia) are redescribed, keyed and figured, including characters of the male and female genitalia of both species. Variation in elytral maculation in D. osculans is discussed and illustrated. The absence of diagnostic differences of D. osculans var. reductus Pic, 1917 from D. osculans is discussed, and the former name is confirmed as a junior subjective synonym of D. osculans. Types of all three names available were studied; a lectotype and paralectotypes are designated for the name D. osculans var. bimaculatus Portevin, 1914. Molecular phylogenetic analysis confirms the genus Diamesus is sister group to the genus Necrodes Leach, 1815, and D. osculans and D. bimaculatus are two, well supported clades. Detailed data on the distribution of D. bimaculatus and D. osculans are presented and mapped. Species distribution models for both species were created and interpreted. Diamesus osculans is reported for the first time from India: Uttarakhand, China: Anhui, Hainan, Hunan, Jiangxi, Shaanxi and Zhejiang Provinces, and Australia: Victoria; it is also recently confirmed from Taiwan, being sympatric in distribution there with D. bimaculatus. Available data on the ecology and seasonality of both species of Diamesus are also discussed.

The Influence of Clay Structures to the Hygrothermal Component of the Indoor Environment.

In this article, research on the sorption properties of clay materials in comparison with commonly used building materials is published. The topic is mainly focused on the dynamic sorption properties and their influence on the relative humidity in the indoor environment. The results of comparisons of clay structures, rammed earth panels, clay plaster, and unburned bricks, with commonly used building materials, concrete, lime plaster, and gypsum board are examined. Statistically evaluated results in the form of confidence intervals are presented and the rate of dynamic sorption is analyzed. It is clear from the results that clay materials have a positive effect on the rapid adsorption and desorption of air moisture in the interior of buildings. However, there are many variables, band not every clay material has such excellent sorption properties.

Degradation of antibacterial 1-octylpyrrolidin-2-one by bacterial pairs isolated from river water and soil.

The study of bacterial degradation of 1-octylpyrrolidin-2-one (NOP) by river water and soil bacteria was the main aim of the research. Although the compound demonstrated bacteriostatic as well as bactericidal effects against Gram-positive and certain Gram-negative bacteria at concentrations ranging from 100 to 1000 mg L-1, its concentration of 100 mg L-1 was successfully degraded by microbial communities of both river water and alluvial soil; removal efficiencies reached 87.2 and 88.4% of dissolved organic carbon, respectively. Isolation of the strains responsible for the process showed that bacterial degradation was initiated by the octane-utilising bacteria of the genus Phenylobacterium, which used four carbon atoms of the NOP octyl chain and oxidised terminal carbon atom of the remaining chain. The structure of the intermediate produced by phenylobacteria was elucidated following the results obtained from the detailed electrospray mass spectrometry (ESI-MS) analysis; these experiments showed that it is a 4-(2-oxopyrrolidin-1-yl)butanoic acid. This intermediate was further degraded by other bacterial members of appropriate microbial communities, namely Bordetella petrii and Arthrobacter sp. Further tests proved that these bacteria were able to assimilate the nitrogen atom of the lactam ring and thus complete the degradation process.

Description of larval morphology and phylogenetic relationships of Heterotemna tenuicornis (Silphidae).

Providing clear and detailed morphological descriptions of endemic species in limited areas enables new knowledge of their biology and ecology to be obtained through citizen science. This information can be further used for their protection. Our study presents the first morphological description of the larvae of all three instars of Heterotemna tenuicornis (Brullé, 1836), an endemic species of the Canary Islands that, together with H. britoi García & Pérez, 1996 and H. figurata (Brullé, 1839), belongs to the peculiar genus Heterotemna Wollaston, 1864. Furthermore, we present the first sequences of two mitochondrial genes (COI, 16S) obtained from larval specimens, and cross reference them with sequences from an adult specimen. Phylogenetic analysis of molecular data placed the genus Heterotemna within the genus Silpha Linnaeus, 1758, suggesting paraphyly of Silpha. In our study, we underline the importance of using a combination of morphological description and molecular data, that can be used for barcoding developmental stages which could not otherwise be definitely associated.

Developmental Biology of Forensically Important Beetle, Necrophila (Calosilpha) brunnicollis (Coleoptera: Silphidae).

The postmortem interval (PMI) estimation, in cases where the body was discovered in an advanced stage of decomposition, is predominantly based on entomological evidence. However, very few forensically important species are sufficiently known in detail to allow a practical application. One of them is the carrion beetle, Necrophila (Calosilpha) brunnicollis (Kraatz, 1877). Its development from egg to adulthood was studied under a range of ecologically relevant constant temperatures to find parameters of thermal summation models. Developmental sexual dimorphism and the presence of developmental rate isomorphy were investigated. Herein we present the lower developmental thresholds and sum of effective temperatures for all developmental stages of N. brunnicollis (egg, first-third larval instar, postfeeding stage, and pupae). We did not find any evidence of developmental sexual dimorphism nor was the presence of developmental rate isomorphy confirmed. Our results present the first thermal summation model of the East Asian carrion beetle that can be used for the PMI estimation.

emOiceoptoma/em emtangi/em (Coleoptera: Silphidae: Silphinae), a new species of carrion beetle from eastern China.

A new species, Oiceoptoma tangi Sommer, Ruzicka, Nishikawa Schneider, new species, from Zhejiang Province, China, is described and illustrated. The new species is closely related to O. subrufum (Lewis, 1888), distributed in Central and North-eastern China, the Korean Peninsula, Far East of Russia and Japan, and to O. nigropunctatum (Lewis, 1888), endemic to Japan. All three species are keyed, and distribution maps of all three species are presented.

Examining aerobic degradation of chloroethenes mixture in consortium composed of Comamonas testosteroni RF2 and Mycobacterium aurum L1.

An environmental isolate Comamonas testosteroni RF2 has been previously described to cometabolize trichloroethene (TCE), 1,2-cis-dichloroethene (cDCE), 1,2-trans-dichloroethene (tDCE), and 1,1-dichloroethene (1,1DCE) when grown on phenol and lactate sodium. In this study, three vinyl chloride (VC) degrading strains, Mycobacterium aurum L1, Pseudomonas putida PS, and Rhodococcus ruber Sm-1 were used to form consortia with the strain RF2 in terms to achieve the removal of VC along with above-mentioned chloroethenes. Degradation assays were performed for a binary mixture of cDCE and VC as well as for a mixture of TCE, all DCEs and VC. The consortium composed of C. testosteroni RF2 and M. aurum L1 showed to be the most efficient towards the removal of cDCE (6.01 mg L-1) in the binary mixture with VC (10 mg L-1) and was capable of efficiently removing chloroethenes in the mixture sample at the initial concentrations of 116 µg L-1 for TCE, 662 µg L-1 for cDCE, 42 µg L-1 for tDCE, 16 µg L-1 for 1,1DCE, and 7 mg L-1 for VC with a removal efficiency of nearly 100% for all of the compounds. Although complete removal of VC took a significantly longer time than the removal of other chloroethenes, the consortium composed of C. testosteroni RF2 and M. aurum L1 displayed strong bioremediation potential for aquifers with downstream contamination characterized by the presence of less chlorinated ethenes.

Developmental Models of the Forensically Important Carrion Beetle, Thanatophilus sinuatus (Coleoptera: Silphidae).

Carrion beetles of genus Thanatophilus (Leach, 1815) are an important group of necrophagous insects, with great potential for forensic entomology in temperate zones of Africa, America, Asia, and Europe. Developmental models for majority of Thanatophilus species remain unknown. In this study, we will provide new thermal summation models for all the developmental stages of Thanatophilus sinuatus (Fabricius, 1775), one of the most abundant and widespread species of the genus. The beetles were bred at seven different constant temperatures, and developmental time was measured for each developmental stage (egg, three larval instars, postfeeding stage, and pupa). Temperature-sex influence was tested, and thermal summation constants were calculated to be used for postmortem interval estimation during criminal investigations.

Inhibition of selenoprotein synthesis by Zika virus may contribute to congenital Zika syndrome and microcephaly by mimicking SELENOP knockout and the genetic disease PCCA.

Selenium status plays a major role in health impacts of various RNA viruses. We previously reported potential antisense interactions between viral mRNAs and host mRNAs encoding isoforms of the antioxidant selenoprotein thioredoxin reductase (TXNRD). Here, we examine possible targeting of selenoprotein mRNAs by Zika virus (ZIKV), because one of the most devastating outcomes of ZIKV infection in neonates, microcephaly, is a key manifestation of Progressive Cerebello-Cerebral Atrophy (PCCA), a genetic disease of impaired selenoprotein synthesis. Potential antisense matches between ZIKV and human selenoprotein mRNAs were identified computationally, the strongest being against human TXNRD1 and selenoprotein P (SELENOP), a selenium carrier protein essential for delivery of selenium to the brain. Computationally, ZIKV has regions of extensive (~30bp) and stable (ΔE < -50kcal/mol) antisense interactions with both TXNRD1 and SELENOP mRNAs. The core ZIKV/SELENOP hybridization was experimentally confirmed at the DNA level by gel shift assay using synthetic oligonucleotides. In HEK293T cells, using Western blot probes for SELENOP and TXNRD1, ZIKV infection knocked down SELENOP protein expression almost completely, by 99% (p<0.005), and TXNRD1 by ~90% (p<0.05). In contrast, by RT-qPCR, there was no evidence of significant changes in SELENOP and TXNRD1 mRNA levels after ZIKV infection, suggesting that their knockdown at the protein level is not primarily a result of mRNA degradation. These results suggest that knockdown of SELENOP and TXNRD1 by ZIKV in fetal brain, possibly antisense-mediated, could mimic SELENOP knockout, thereby contributing to neuronal cell death and symptoms similar to the genetic disease PCCA, including brain atrophy and microcephaly.

Selenium-Dependent Read Through of the Conserved 3'-Terminal UGA Stop Codon of HIV-1 nef.

The HIV-1 nef gene terminates in a 3'-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) [1]. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3'-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by >100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.

Direct Detection of Conserved Viral Sequences and Other Nucleic Acid Motifs with Solid-State Nanopores.

The rapid and reliable recognition of nucleic acid sequences is essential to a broad range of fields including genotyping, gene expression analysis, and pathogen screening. For viral detection in particular, the capability is critical for optimal therapeutic response and preventing disease transmission. Here, we report an approach for detecting identifying sequence motifs within genome-scale single-strand DNA and RNA based on solid-state nanopores. By designing DNA oligonucleotide probes with complementarity to target sequences within a target genome, we establish a protocol to yield affinity-tagged duplex molecules the same length as the probe only if the target is present. The product can subsequently be bound to a protein chaperone and analyzed quantitatively with a selective solid-state nanopore assay. We first use a model DNA genome (M13mp18) to validate the approach, showing the successful isolation and detection of multiple target sequences simultaneously. We then demonstrate the protocol for the detection of RNA viruses by identifying and targeting a highly conserved sequence within human immunodeficiency virus (HIV-1B).