Development of a multi-excitation fluorescence (MEF) imaging method to improve the information content of benthic coral reef surveys

Benthic surveys are a key component of monitoring and conservation efforts for coral reefs worldwide. While traditional image-based surveys rely on manual annotation of photographs to characterise benthic composition, automatic image annotation based on computer vision is becoming increasingly common. However, accurate classification of some benthic groups from reflectance images presents a challenge to local ecologists and computers alike. Most coral reef organisms produce one or a combination of fluorescent pigments, such as Green Fluorescent Protein (GFP)-like proteins found in corals, chlorophyll-a found in all photosynthetic organisms, and phycobiliproteins found in red macroalgae, crustose coralline algae (CCA) and cyanobacteria. Building on the potential of these pigments as a target for automatic image annotation, we developed a novel imaging method based on off-the-shelf components to improve classification of coral and other biotic substrates using a multi-excitation fluorescence (MEF) imaging system. We used RGB cameras to image the fluorescence emission of coral and algal pigments stimulated by narrow-waveband blue and green light, and then combined the information into three-channel pseudocolour images. Using a set of a priori rules defined by the relative pixel intensity produced in different channels, the method achieved successful classification of organisms into three categories based on the dominant fluorescent pigment expressed, facilitating discrimination of traditionally problematic groups. This work provides a conceptual foundation for future technological developments that will improve the cost, accuracy and speed of coral reef surveys.

     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

Tau regulates the localization and function of End‐binding proteins 1 and 3 in developing neuronal cells

The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU?/? neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation.

     

On the Interplay of Telomeres,Nevi and the Risk of Melanoma

The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.     

Comparative analysis reveals amino acids critical for anticancer activity of peptide CIGB‐552

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB‐552, a novel cell‐penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor‐bearing mice. Studies of protein–peptide interactions have shown that COMMD1 protein is a major mediator of CIGB‐552 antitumor activity. Furthermore, a typical serine‐protease degradation pattern for CIGB‐552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB‐552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB‐552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF‐7 cells. None of the analyzed metabolites proved to be as effective as CIGB‐552 in promoting apoptosis in MCF‐7. Taking into account these results, it seemed important to examine their cell‐penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding‐dependent process, is impaired as well as metabolite–COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB‐552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structure of a methyltransferase from a no-known-vector Flavivirus

Presently known flaviviruses belong to three major evolutionary branches: tick-borne viruses, mosquito-borne viruses and viruses with no known vector. Here we present the crystal structure of the Yokose virus methyltransferase at 1.7 Å resolution, the first structure of a methyltransferase of a Flavivirus with no known vector. Structural comparison of three methyltransferases representative of each of the Flavivirus branches shows that fold and structures are closely conserved, most differences being related to surface loops flexibility. Analysis of the conserved residues throughout all the sequenced flaviviral methyltransferases reveals that, besides the central cleft hosting the substrate and cofactor binding sites, a second, almost continuous, patch is conserved and points away from active site towards the back of the protein. The high level of structural conservation in this region could be functional for the methyltransferase/RNA interaction and stabilization of the ensuing complex.     

Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus

The mRNA-capping process starts with the conversion of a 5′-triphosphate end into a 5′-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5′-5′ phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2′OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2′O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, ^N7MeGpppG, and ^N7MeGpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2′O-methyltransferase activities.     

Congenital hypothyroidism and pericardial effusion

A group of infants, affected by congenital hypothyroidism diagnosed through the neonatal screening program, was investigated with echocardiography to detect the presence of pericardial effusion. We studied the relationship between the effusion and the etiology of hypothyroidism, established through thyroid scintiscanning. Our data show a high prevalence of effusion in hypothyroid patients, without other clinical signs of cardiac involvement as well as a relationship between the etiology of hypothyroidism and the presence of effusion. This seems to be much more frequent in those forms which can imply a more severe hormonal defect, particularly during fetal life (agenesis/dyshormonogenesis). Furthermore, the high prevalence of pericardial effusion suggests to start the L-T4 replacement therapy with lower dosages as commonly advised, in order to avoid a cardiac involvement.     

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.     

Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells

Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 degrees C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.