Laser diode driver on a programmable system on a chip.

A comprehensive solution of a laser diode (LD) driver with temperature control using a programmable system on a chip is proposed as an alternative to dedicated devices. A digital proportional integral differential controller is used for regulating the LD temperature. The light-current characteristics and frequency response of the LD are measured using an external photodiode and interrogated by an integrated transimpedance amplifier. The 0.084% stability, 2 kHz bandwidth, and 0.11% full scale current error are demonstrated. The LD overshoot protection is digitally implemented, providing a soft start. While the circuit is initially optimized for a 3 W infrared LD, instructions are provided for fine-tuning the design according to specific LD requirements.

Haplosporidium pinnae Parasite Detection in Seawater Samples.

In this study, we investigated the presence of the parasite Haplosporidium pinnae, which is a pathogen for the bivalve Pinna nobilis, in water samples from different environments. Fifteen mantle samples of P. nobilis infected by H. pinnae were used to characterize the ribosomal unit of this parasite. The obtained sequences were employed to develop a method for eDNA detection of H. pinnae. We collected 56 water samples (from aquaria, open sea and sanctuaries) for testing the methodology. In this work, we developed three different PCRs generating amplicons of different lengths to determine the level of degradation of the DNA, since the status of H. pinnae in water and, therefore, its infectious capacity are unknown. The results showed the ability of the method to detect H. pinnae in sea waters from different areas persistent in the environment but with different degrees of DNA fragmentation. This developed method offers a new tool for preventive analysis for monitoring areas and to better understand the life cycle and the spread of this parasite.

An incubation water eDNA method for a non-destructive rapid molecular identification of Pinna nobilis and Pinna rudis bivalve juveniles.

The pen shell Pinna nobilis is critically endangered due to a disease that has affected all open water populations since late 2016. Collection of early spats is considered a fundamental step for pen shell conservation. However, the identification between P. nobilis and P. rudis juveniles by morphology is a very difficult task. Furthermore, due to the small size of juveniles and high sensitivity to handling, the sampling for this purpose must not damage individuals. As a consequence, the application of molecular techniques for conservation strategies to identify threatened and endangered bivalve species is every day more and more necessary. In this study, we present the development of a multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples. Using species-specific primers, designed in the rRNA16S and rRNA12S mitochondrial genes, identification of species was obtained by cellular or extracellular DNA dissolved in water and differentiated based on the size of the amplified DNA fragments. • Development of a molecular multiplex-PCR procedure for the rapid identification of two Pinna species from eDNA water samples • Using specie-specific primers, the different species can be differentiated basing on the size of the amplified DNA fragments • This technique removes many of the limitations commonly associated with sampling of threatened and endangered juvenile bivalves for conservation strategies (sampling does not damage individuals).

p63 in Mytilus galloprovincialis and p53 family members in the phylum Mollusca.

Genes of the p53 family are known to be critical regulators of the cell cycle. They have already been established as possible biomarkers. Elaborate regulation mechanisms result in numerous cDNA and protein isoforms being expressed from each gene of the p53 family. Their similarity caused an often misleading nomenclature in non-vertebrate species. The aim of the present work is a clarification of the nomenclature of molluscan p53 family sequences, an essential prerequisite for reliable interpretation of gene expression and protein function studies. Here, we report five partial cDNA and one partial genomic p63 sequences, all originating from two Mytilus galloprovincialis individuals. DNA, deduced protein sequences, and the exon/intron architecture were analyzed and compared to p53, p63 and p73 sequences from other organisms. Along with our sequences, we analyzed all similar molluscan sequences found in the GenBank database. The analysis showed our cDNA sequences code for the TAp63gamma isoform of the p63 protein, and identified all other molluscan p53 family sequences as p63 genes or their expression isoforms. Our results also indicate p63 as the ancestral gene of the p53 family as well as the only gene of the family present in non-chordate metazoan species.

Adriatic coast as a microcosm for global genotoxic marine contamination--a long-term field study.

Global changes in the marine environment and the continuing disposal of genotoxic xenobiotics are increasing the importance of environmental pollution monitoring and of biomonitoring programs. Current approaches focus on investigations at regional and local levels in an attempt to precisely define the nature and extent of any potential environmental crisis. We have initiated, for the first time, a long-term biomonitoring program focusing on the Croatian coast of the Adriatic Sea to contribute to a more detailed understanding of marine genotoxic effects using the mussels Mytilus galloprovincialis Lam., collected along the eastern Adriatic coast over a period of five years (1998-2002), as a key test organism. The integrity of DNA in its gill homogenate was examined by the Fast Micromethod. The strand scission factor (SSF) values, as a measure of DNA integrity, DNA damage or incomplete repair have been used for the ranking of sampling sites with respect to significant genotoxic stress due to the influence or effects of genotoxic xenobiotics. The region of Split (Kastela Bay) proved to be the area with the heaviest load of genotoxic agents. The investigation of harmful effects in the ecosystem based on biomonitoring of genetic and other agents, not only on local levels but also on a wider scale, is considered as an important step in marine environmental management.

Seawater quality along the Adriatic coast, Croatia, based on toxicity data.

The potential toxicity of organic extracts from 12 seawater samples from each of 24 sampling sites, collected during 1999-2001 along the Adriatic coast, Croatia, was analyzed with the Microtox toxicity bioassay. The results were consistent with the usefulness of Microtox for the detection of accidental toxic events. To determine the water quality of selected areas, cluster analysis for discrimination between groups with similar toxicity load and water quality index as a base for the ranking of sampling sites was introduced. Based on our experimental data, five classes of the quality index were defined, and so areas were ranked in five categories (excellent, good, fair, poor, and very poor) according to their potential toxic influence. The water quality of selected sites for the potential toxicity of organic extracts could be described as excellent at one sampling site, good at 15 sampling sites, and fair at eight sampling sites. Poor and very poor seawater quality was not detected.

Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia.

Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants.

Application of alkaline elution, Fast Micromethod and flow cytometry in detection of marine contamination.

DNA damage is an inescapable aspect of life in the biosphere. The presented investigations were an attempt to examine the response of a DNA damage as a biomarker of environmental quality in the mussels Mytilus galloprovincialis sampled at differently contaminated areas of Istrian coast, Northern Adriatic. The investigations were performed in order to get information about the genotoxic risk for marine organisms exposed to mixed environmental pollution, as well as the information about the presence of unknown mixture of genotoxic contaminants in the marine environment. Types of DNA damage detected are alkali-labile sites and single-strand breaks measured by Fast Micromethod, interstrand cross-links and DNA protein cross-links by alkaline filter elution and cell cycle disturbation by flow cytometry. The applicability of all three methods for marine quality control is discussed.

DNA damage and apoptosis in the mussel Mytilus galloprovincialis.

The effects of known genotoxic substances (4-nitroquinoline-N-oxide, benzo[a]pyrene, teniposide, etoposide, cycloheximide, tributyltin) on human cells (FLC, HL-60) and on mussels were investigated. The correlations between formation of DNA strand breaks and DNA fragmentation characteristic for the process of apoptosis were estimated. Strand breaks induced by 4-nitroquinoline-N-oxide and benzo[a]pyrene did not correlate with DNA fragmentation detected in the process of apoptosis. Induction of internucleosomal DNA fragmentation in HL-60 cells was initiated by teniposide, etoposide and tributyltin, while in the gills of mussels this was detected only with tributyltin. Levels of DNA strand breaks in natural mussel populations, living at locations under the influence of urban and industrial wastes, do not mirror the apoptotic processes.