Atomic Force Microscope nanolithography on chromosomes to genrate single-cell genetic probes

Abstract Background Chromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level. Results We report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei. Conclusions Atomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.</p

\u3ci\u3eScaphanocephalus\u3c/i\u3e-Associated Dermatitis as the Basis for Black Spot Disease in Acanthuridae of St. Kitts, West Indies

Acanthurus spp. of St. Kitts and other Caribbean islands, including ocean surgeonfish A. bahianus, doctorfish A. chirurgus, and blue tang A. coeruleus, frequently show multifocal cutaneous pigmentation. Initial reports from the Leeward Antilles raised suspicion of a parasitic etiology. The aim of this study was to quantify the prevalence of the disease in St. Kitts’ Acanthuridae and describe its pathology and etiology. Visual surveys demonstrated consistently high adjusted mean prevalence at 3 shallow reefs in St. Kitts in 2017 (38.9%, 95% CI: 33.8−43.9) and 2018 (51.5%; 95% CI: 46.2−56.9). There were no differences in prevalence across species or reefs, but juvenile fish were less commonly affected than adults. A total of 29 dermatopathy-affected acanthurids were sampled by spearfishing for comprehensive postmortem examination. Digenean metacercariae were dissected from \u3c1 mm cysts within pigmented lesions. Using partial 28S rDNA sequence data they were classified as Family Heterophyidae, members of which are commonly implicated in black spot disease of other fishes. Morphological features of the parasite were most typical of Scaphanocephalus spp. (Creplin, 1842), and 2 genetic profiles were obtained suggesting more than 1 digenean species. Histologically, pigmented lesions had mild chronic perivascular dermatitis and increased melanophores and melanin density, often centered on encysted digenean metacercariae. In 1 affected A. chirurgus, similar metacercariae were histologically identified in skeletal and cardiac muscle. Further research is needed to clarify impact on host fitness, establish the number of heterophyid digenean species that cause black spots on Caribbean fishes and to determine the intermediate and definitive host species

Novel atrazine-binding biomimetics inspired to the D1 protein from the photosystem II of Chlamydomonas reinhardtii

Biomimetic design represents an emerging field for improving knowledge of natural molecules, as well as to project novel artificial tools with specific functions for biosensing. Effective strategies have been exploited to design artificial bioreceptors, taking inspiration from complex supramolecular assemblies. Among them, size-minimization strategy sounds promising to provide bioreceptors with tuned sensitivity, stability, and selectivity, through the ad hoc manipulation of chemical species at the molecular scale. Herein, a novel biomimetic peptide enabling herbicide binding was designed bioinspired to the D1 protein of the Photosystem II of the green alga Chlamydomonas reinhardtii. The D1 protein portion corresponding to the QB plastoquinone binding niche is capable of interacting with photosynthetic herbicides. A 50-mer peptide in the region of D1 protein from the residue 211 to 280 was designed in silico, and molecular dynamic simulations were performed alone and in complex with atrazine. An equilibrated structure was obtained with a stable pocked for atrazine binding by three H-bonds with SER222, ASN247, and HIS272 residues. Computational data were confirmed by fluorescence spectroscopy and circular dichroism on the peptide obtained by automated synthesis. Atrazine binding at nanomolar concentrations was followed by fluorescence spectroscopy, highlighting peptide suitability for optical sensing of herbicides at safety limits

Rapporto sull’attività 29 Giugno – 6 Luglio 2013

In questo lavoro viene descritta la campagna geofisica e idro-oceanografica ‘GARGANO2013’ effettuata grazie ad una collaborazione tra INGV e Marina Militare Italiana nell’ambito di un accordo di collaborazione denominato CONAGEM (Coordinamento Nazionale per la Geofisica Marina). Tale accordo, siglato nel 2005 fra i principali enti pubblici di ricerca che operano in mare, prevede infatti la possibilità di attuare congiuntamente campagne di ricerca marine condividendone dati e risultati. La campagna è stata organizzata con la finalità di raccogliere quanti più possibili elementi utili a caratterizzare l’area marina del Gargano sotto un profilo ambientale predisponendone un quadro di riferimento per successive attività di monitoraggio delle fenomenologie presenti nell’area di indagine. In quest’ottica, l’obiettivo prefissato era la mappatura di eventuali affioramenti di acque dolci in mare. Contestualmente, è stata eseguita la sperimentazione di prototipi strumentali per future applicazioni a bordo nave: in particolare, sono state eseguite misure di tipo accelerometrico al fine di caratterizzare le sollecitazioni dinamiche cui sono sottoposti gli strumenti a bordonave ottenendo così utili informazioni per lo sviluppo di nuove tecnologie quali piattaforme inerziali per uso scientifico e strumentazione per prospezioni gravimetriche da utilizzare su nave

Diseño de un instrumento portátil para aplicaciones ambientales por mediciones amperométricas sobre material biológico

El instrumento portátil optimizado para las medidas  de amperometria, en el monitoreo de  materiales bioactivos ha sido diseñado, fabricado y probado. Expresamente ha sido diseñado, para funcionar con una amplia gama de bio-muestras foto-activas. La cámara  de medición del instrumento; destaca dos tipos de fuentes ópticas para detectar la actividad fotosintética de plantas (p. ej. spinacia oleracea) y microorganismos (p. ej. algas y cyanobacteria). En la cámara son inseridos los electrodos serigrafiados para medir la corriente fotogenerada, ademas cuenta con un sistema de flujo para el transporte del electrólito. La transferencia fotosintética de electrones, es activada por dos LEDs (470nm y 660nm de emisión), para permitir varias longitudes de onda de excitación para utilizarlos con diversos materiales biológicos. El objetivo de la aplicacion, es en campos como agroalimentario, farmacéutico y biomédico. Este Artículo describe algunas de las posibles aplicaciones ambientales ABSTRACT A portable instrument performing amperometric measurements for monitoring bioactive materials has been designed, manufactured and tested. It has been specifically designed to operate with a wide range of photoactive biosamples. The sensing chamber in the instrument features two different optical sources to detect the photosynthetical activity of plants (i.e. spinacia oleracea) and microorganisms (i.e. algae and cyanobacteria). The chamber is provided with screen-printed electrodes to measure the photogenerated current and with a fluidic system for the electrolyte transport. Photosynthetic electron transfer is activated by two LEDs (470nm and 660nm emission) in order to enable various excitation wavelengths and match several different biological materials. Target applications belong to the agro-food, pharmaceutical and biomedical fields. This paper describes some possible environmental application

Carbonaceous aerosol in polar areas: First results and improvements of the sampling strategies

While more and more studies are being conducted on carbonaceous fractions—organic carbon (OC) and elemental carbon (EC)—in urban areas, there are still too few studies about these species and their effects in polar areas due to their very low concentrations; further, studies in the literature report only data from intensive campaigns, limited in time. We present here for the first time EC–OC concentration long-time data records from the sea-level sampling site of Ny-Ålesund, in the High Arctic (5 years), and from Dome C, in the East Antarctic Plateau (1 year). Regarding the Arctic, the median (and the interquartile range (IQR)) mass concentrations for the years 2011–2015 are 352 (IQR: 283–475) ng/m3 for OC and 4.8 (IQR: 4.6–17.4) ng/m3 for EC, which is responsible for only 3% of total carbon (TC). From both the concentration data sets and the variation of the average monthly concentrations, the influence of the Arctic haze on EC and OC concentrations is evident. Summer may be interested by high concentration episodes mainly due to long-range transport (e.g., from wide wildfires in the Northern Hemisphere, as happened in 2015). The average ratio of EC/OC for the summer period is 0.05, ranging from 0.02 to 0.10, and indicates a clean environment with prevailing biogenic (or biomass burning) sources, as well as aged, highly oxidized aerosol from long-range transport. Contribution from ship emission is not evident, but this result may be due to the sampling time resolution. In Antarctica, a 1 year-around data set from December 2016 to February 2018 is shown, which does not present a clear seasonal trend. The OC median (and IQR) value is 78 (64–106) ng/m3; for EC, it is 0.9 (0.6–2.4) ng/m3, weighing for 3% on TC values. The EC/OC ratio mean value is 0.20, with a range of 0.06–0.35. Due to the low EC and OC concentrations in polar areas, correction for the blank is far more important than in campaigns carried out in other regions, largely affecting uncertainties in measured concentrations. Through the years, we have thus developed a new sampling strategy that is presented here for the first time: samplers were modified in order to collect a larger amount of particulates on a small surface, enhancing the capability of the analytical method since the thermo-optical analyzer is sensitive to carbonaceous aerosol areal density. Further, we have recently coupled such modified samplers with a sampling strategy that makes a more reliable blank correction of every single sample possible

Rapporto sull’attività 29 Giugno – 6 Luglio 2013

In questo lavoro viene descritta la campagna geofisica e idro-oceanografica ‘GARGANO2013’ effettuata grazie ad una collaborazione tra INGV e Marina Militare Italiana nell’ambito di un accordo di collaborazione denominato CONAGEM (Coordinamento Nazionale per la Geofisica Marina). Tale accordo, siglato nel 2005 fra i principali enti pubblici di ricerca che operano in mare, prevede infatti la possibilità di attuare congiuntamente campagne di ricerca marine condividendone dati e risultati. La campagna è stata organizzata con la finalità di raccogliere quanti più possibili elementi utili a caratterizzare l’area marina del Gargano sotto un profilo ambientale predisponendone un quadro di riferimento per successive attività di monitoraggio delle fenomenologie presenti nell’area di indagine. In quest’ottica, l’obiettivo prefissato era la mappatura di eventuali affioramenti di acque dolci in mare. Contestualmente, è stata eseguita la sperimentazione di prototipi strumentali per future applicazioni a bordo nave: in particolare, sono state eseguite misure di tipo accelerometrico al fine di caratterizzare le sollecitazioni dinamiche cui sono sottoposti gli strumenti a bordonave ottenendo così utili informazioni per lo sviluppo di nuove tecnologie quali piattaforme inerziali per uso scientifico e strumentazione per prospezioni gravimetriche da utilizzare su nave.Marina Militare ItalianaPublished1-283A. Ambiente MarinoN/A or not JCRope