Month: June 2020

Historically they arrived first, ranging from simple majority fraction to information entropy and entropy related methods, to full-blown statistical estimation of the mutability of residues leading to the observed set of sequences. Such methods work well in detecting the folding core of a protein, the catalytic site of an enzyme, and somewhat less reliably, the protein-protein interfaces shared by all homologues. Their performance is affected more strongly by the preprocessing stage, then by the choice of method itself. The specialization of duplicated genes is the necessary condition for their parallel existence, and the methods to detect it on the protein level followed shortly. Several major ways of treating this problem have been put forth, differing mainly in the way they handle. The first issue has been dealt with by taking the classification as an input, by using the similarity tree as the classification generator, or by adopting a midway solution in which the tree is provided by the application, but the relevant division into subtrees is decided on by the user. In this work, we would like to put some emphasis on the way an evolutionary model is built into a specificity scoring function. As an example, a popularly quoted evolutionary trace method, ET, in its original formulation assumes that a functionally important position will be completely conserved in each of the compared groups of sequences, albeit as a different amino acid type. If the groups in question are paralogous, this becomes a very strict model of evolution, in which even after the duplication and specialization event, each gene maintains the same degree of evolutionary pressure at each site. This model appears in the literature in several forms. Conversely, mutual information requires that each group of orthologues adopts a set of evolutionary constraints that are systematically different from those of all other groups, irrespective of the degree of conservation within each group. However, it mirrors “conservatism-of-conservatism” in conditioning the expected behavior in one group, on the behavior in another. Recently, ever more voices appear in the literature, pointing out that the evolutionary behavior in paralogous groups may be completely unrelated. Variously termed “type I functional divergence” or “SB431542 301836-41-9 heterotachy”, this type of behavior has been discussed in genetics literature for at least a decade, and used increasingly in detection of family specific positions on a nucleotide or peptide sequence. Finding the “type I – type II” terminology somewhat lacking in descriptive power, we use the term “determinants” for the positions that are conserved in one group, but evolve at various rates across paralogues, and “discriminants” for the positions that vary at comparable low rates across all groups. A determinant position, then, is a property of a single group, while a discriminant is a property of the family as a whole.

Thus, cultureindependent methods allow to overcome biases associated to the culturing step. The detection of microbial populations from total DNA or RNA extracted directly from food matrices can give a more realistic and reliable “picture” of cheese microbiota. In order to monitor the presence and viability of L. lactis throughout cheese manufacturing and ripening, a highly selective qPCR protocol was optimized. The detection of L. lactis with respect to other LAB species, which normally colonize ripened cheeses, was reached by selective primer design on tuf gene codyfing a GTP binding protein and widespread in eubacteria genomes. Tuf gene has been generally recognized as a housekeeping gene ; moreover, its stability was confirmed by studying its expression throughout L. lactis growth curve. SYBR green fluorescent chemistry was chosen and good results were obtained, in terms of specificity, correlation coefficient and efficiency, by increasing the stringency of the thermal cycle and using primers in unbalanced concentration. In particular, the high annealing temperature, used in qPCR and RT-qPCR protocols, allowed the specific detection of L. lactis and no fluorescent signal was detected when the protocol was applied to the other LAB species. Thus, tuf gene represented a suitable target for the specific detection and quantification of L. lactis as also highlighted by other authors. Moreover, the efficiency of the protocols was improved by the choice of nucleic acid extraction protocols specifically designed for the treatment of fatty matrices, highlighting, once again, how this step heavily influence the performance of the subsequent amplification. The high quality of the extracted RNA and the set amplification conditions allowed to obtain standard curves with a good linearity range covering 6 orders of magnitude, from 102 to 108 CFU/g. Other authors optimized qPCR protocols to detect L. lactis in milk, under simulated conditions of cheese manufacture, in ultrafiltered milk cheese models and in the manufacturing of raw milk soft cheeses. The only study dealing with the monitoring of active population of L. lactis in cheese Y-27632 dihydrochloride ripening by RT-qPCR has been focused on Cheddar cheese, for which undefined starter cultures containing L. lactis subsp. lactis and L. lactis subsp. cremoris are commonly used. In particular, the authors evaluated the impact of milk heat treatments and ripening temperatures on lactococcal starter and NSLAB throughout maturing of Cheddar cheese and the results showed that lactococci remained dominant throughout the ripening process. The results presented in this study, however, do not shed light into the possible contribution of L. lactis in terms of organoleptic characteristics of the final cheese product. Thus, as future prospective, it will be important to investigate the role, in terms of metabolic activities, of this microorganism during cheese ripening. In particular, it will be interesting to understand which L. lactis functions are being carried out in each specific phase of the production, with the final aim of improving technological processes and cheese quality.

Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including cheese samples were no L. lactis was found by RT-qPCR. In these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. Probably, lactococci are able to grow on M17 medium when they are abundant and not stressed, as for example during milk and curd fermentation. Differently, during the ripening process, it is known that NSLAB increase in number and prevail on lactococcal populations, which are often out-competed by the numerically more abundant lactobacilli. Nevertheless, in this work, a few isolates were identified as L. lactis by His-PCR. They were obtained from eight cheese samples with loads higher than 107 CFU/g, detected by RT-qPCR, except for two samples characterized by values of 104 and 106 CFU/g. These data could be explained with the relative high abundance of L. lactis in these cheeses and, thus, its capability to compete with the rest of microbiota and multiply on synthetic media. Currently, M17 is the medium mainly used for lactococci cultivation, but new formulations for the isolation of LAB from cheese have been recently studied as, for example, cheese agar, which was used to recover minority populations from milk, whey starter and fresh curd of Parmigiano Reggiano, hardly estimable on traditional media. Thus, as future prospective, for a more reliable and effective recovery of lactococci, in particular L. lactis, during cheese ripening, the optimization and formulation of specific nutritional conditions should be better investigated. The absence or low abundance of L. lactis isolates on M17 medium, support the thesis hypothesized by other authors that L. lactis starter populations are mainly present in VNC state during cheese ripening and, for this reason, culturedependent methods are not able to detect their presence and have to be complemented with direct analysis in cheese. These considerations can be especially corroborated from the results obtained in eight cheese samples where the difference, in terms of microbial load, between RT-qPCR and plating data, was lower than 102 CFU/g and, thus, the absence of L. lactis growth, on M17, could not be justifiable with the prevalence of NSLAB. For some of the cheeses analysed, experiments were performed in order to “resuscitate” L. lactis VNC cells and preliminary results highlighted that different AG-013736 carbon sources, in cultural media, affect differently their growing ability ; in particular, enrichment in medium with high percentage of glucose seemed to stimulate the attitude of the cells to become culturable again. Recent researches were focused on these aspects and highlighted the presence of VNC L. lactis cells in ripened cheese products. Differently, Flo´rez and colleagues found abundance of L. lactis isolates on M17 from the analysis of Spanish cheese, but they did not specify the distribution of the isolates among milk, curd and cheese samples.

Here we report on our initial successes in overcoming both of these technical challenges. We have developed a technique for injecting reagents into live S. purpuratus larvae, specifically targeting the echinus rudiment: a series of tissues that develop within the larval body, and are fated to form predominantly the oral structures of the urchin juvenile. By injecting rhodaminated dextran, we were able to consistently label structures within the rudiment, providing a technique for visualizing these structures in live larvae. Furthermore, we have manipulated the normal ontogeny of juvenile structures by injecting Vivo-Morpholinos –a class of morpholino oligonucleotides that are designed to cross cell membranes– into various compartments within the rudiment of late stage purple urchin larvae. Specifically, we document an inhibition in growth and elongation of incipient adult spines using vMOs directed against p16 and p58b, two genes known to be involved in skeletal elongation in urchin embryos. We are confident in the specificity of the phenotypes that we report for the vMO rudiment injections for the following reasons: 1) we did not observe CUDC-907 side effects skeletogenic phenotypes in controls, and specifically using the control vMO, in either soaked embryos or injected larvae; 2) we were able to provide evidence that p58b vMOs are effectively eliminating the correct splice variant of the gene and consequently lead to a functional knockdown; 3) the phenotypes that we did observe in embryos with our p16 and p58b vMOs phenocopy previously published results obtained with standard MOs injected into eggs ; and 4) the phenotypes that we observed in our rudiment injections indicated that injected rudiments continued to develop normally after injection, across all treatments – it was only a subset of the skeletal elements specifically in the p16 and p58b vMO treatments that showed inhibited growth. Therefore, our results indicate, for the first time, that morphogenesis of the juvenile sea urchin can be manipulated by morpholino injection. As a corollary, we provide evidence that p16 and p58b are required for normal skeletal elongation during sea urchin juvenile skeletal development, as they are during embryogenesis, a finding consistent with the expression of much of the purple urchin larval skeletogenic regulatory network in juvenile rudiments. The basic protocol that we described should be feasible for most inverted microscopes and injection configurations. One of the most significant challenges that we faced was ascertaining the exact location of the tip of the needle relative to the many tissue layers within the echinus rudiment. The injection microscope we used was not equipped with epi-fluorescence; having that capability would have been preferable, as it would have allowed us to confirm injection location without the need to mount the post-injection larvae under raised cover glass. With our setup, we had the best success when viewing the needle with a partially-closed diaphragm under 2002400x magnification. Watching the liquid being expelled from the needle tip also gave hints as to the location of the injection.

The main reason for this difficulty is that standard genetic manipulations -whether through stable mutagenesis or through factors injected in the egg- generally lead to embryonic phenotypes or lethality, and their larvae would thus be abnormal or not survive to the metamorphic stages under study. More general treatments of larvae likewise cause larval phenotypes independent of their impacts on the structures in larvae fated to form the juvenile: namely, the imaginal discs in holometabolous insects. Also, years of detailed studies in insects led to the ability to culture these imaginal disks in vitro, thus illuminating the previously obscure events of specification and differentiation of presumptive adult structures. We are not aware of any such techniques having been devised for a marine invertebrate, though new functional genomics approaches –that allow the specific manipulation of genes and gene products– should allow more incisive investigations into marine invertebrate larval development, including settlement and metamorphosis. Here we report on our success in targeted manipulation of juvenile development in purple sea urchin larvae. Using a new class of morpholino oligonucleotides that readily cross cell membranes, we describe a technique where we can reliably inject these compounds inside juvenile rudiment tissues of temporarily immobilized late stage S. purpuratus larvae. As a proof of concept, we injected vMOs designed to knock down the expression of p16 and p58b, two genes involved in skeletal elongation in sea urchin embryos. We report a significant decrease in the elongation rate of adult spines in both p16 and p58b-injected larvae when compared to those injected with the control vMO, a carrier control and an uninjected control. Along with our morpholino injections, we also injected rhodaminated dextran as a tracer. These Screening Libraries injections resulted in accumulation of rhodamine into different compartments of the developing rudiment, allowing for visualizations of developing juvenile tissues at a level of detail not previously described. Complex life cycles have evolved repeatedly in animals and non-animals alike. In the majority of coastal marine invertebrates, the most dramatic life cycle transition involves a metamorphosis from a planktonic larva to a benthic adult. This metamorphic transition has profound implications for the ecological stability of marine communities, gene flow among them, and their recovery following disturbance. Furthermore, invertebrate metamorphoses are fascinating developmental events in their own right, where the adults often differ morphologically, behaviorally and ecologically from their corresponding larval forms. Echinoderms such as the purple sea urchin, Strongylocentrotus purpuratus, represent notable examples. Despite the relevance and interest in such transformations across invertebrates and within echinoderms, we have limited mechanistic understanding of the developmental and physiological processes that regulate marine invertebrate metamorphoses. One major impediment to gaining such understanding is the difficulty inherent in manipulating genes in late stage larvae without causing embryonic phenotypes at earlier stages.