Extensive conservation of the proneuropeptide and peptide prohormone complement in mollusks.

As one of the most diverse groups of invertebrate animals, mollusks represent powerful models for neurobiological and developmental studies. Neuropeptides and peptide hormones are a heterogeneous class of signalling molecules involved in chemical communication between neurons and in neuroendocrine regulation. Here we present a fine-grained view of the molluscan neuropeptide and peptide hormone toolkit. Our results expand the distribution of several peptide families (e.g., prokineticin, insulin-related peptides, prohormone-4, LFRFamide) within Lophotrochozoa and provide evidence for an early origin of others (e.g., GNXQN/prohormone-2, neuroparsin). We identified a new peptide family broadly distributed among conchiferan mollusks, the PXRX family. We found the Wnt antagonist dickkopf1/2/4 ortholog in lophotrochozoans and nematodes and reveal that the egg-laying hormone family is a DH44 homolog restricted to gastropods. Our data demonstrate that numerous peptides evolved much earlier than previously assumed and that key signalling elements are extensively conserved among extant mollusks.

Genetic diversity of thermoduric spoilage microorganisms of milk from Brazilian dairy farms.

When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 102 cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk.

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 104 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.

Comparative transcriptomics enlarges the toolkit of known developmental genes in mollusks.

BACKGROUND: Mollusks display a striking morphological disparity, including, among others, worm-like animals (the aplacophorans), snails and slugs, bivalves, and cephalopods. This phenotypic diversity renders them ideal for studies into animal evolution. Despite being one of the most species-rich phyla, molecular and in silico studies concerning specific key developmental gene families are still scarce, thus hampering deeper insights into the molecular machinery that governs the development and evolution of the various molluscan class-level taxa. RESULTS: Next-generation sequencing was used to retrieve transcriptomes of representatives of seven out of the eight recent class-level taxa of mollusks. Similarity searches, phylogenetic inferences, and a detailed manual curation were used to identify and confirm the orthology of numerous molluscan Hox and ParaHox genes, which resulted in a comprehensive catalog that highlights the evolution of these genes in Mollusca and other metazoans. The identification of a specific molluscan motif in the Hox paralog group 5 and a lophotrochozoan ParaHox motif in the Gsx gene is described. Functional analyses using KEGG and GO tools enabled a detailed description of key developmental genes expressed in important pathways such as Hedgehog, Wnt, and Notch during development of the respective species. The KEGG analysis revealed Wnt8, Wnt11, and Wnt16 as Wnt genes hitherto not reported for mollusks, thereby enlarging the known Wnt complement of the phylum. In addition, novel Hedgehog (Hh)-related genes were identified in the gastropod Lottia cf. kogamogai, demonstrating a more complex gene content in this species than in other mollusks. CONCLUSIONS: The use of de novo transcriptome assembly and well-designed in silico protocols proved to be a robust approach for surveying and mining large sequence data in a wide range of non-model mollusks. The data presented herein constitute only a small fraction of the information retrieved from the analysed molluscan transcriptomes, which can be promptly employed in the identification of novel genes and gene families, phylogenetic inferences, and other studies using molecular tools. As such, our study provides an important framework for understanding some of the underlying molecular mechanisms involved in molluscan body plan diversification and hints towards functions of key developmental genes in molluscan morphogenesis.

Screening of inbred popcorn lines for tolerance to low phosphorus.

Increasing phosphorus use efficiency in agriculture is essential for sustainable food production. Thus, the aims of this study were: i) to identify phosphorus use efficiency (PUE) in popcorn lines during the early plant stages, ii) to study the relationship between traits correlated with PUE, and iii) to analyze genetic diversity among lines. To accomplish this, 35 popcorn lines from Universidade Estadual de Maringá breeding program were studied. The experiment was conducted in a growth chamber using a nutrient solution containing two concentrations of phosphorus (P): 2.5 µM or low P (LP) and 250 µM or high P (HP). After 13 days in the nutrient solution, root morphology traits, shoot and root dry weight, and P content of the maize seedlings were measured. A deviance analysis showed there was a high level of genetic variability. An unweighted pair group method with arithmetic mean (UPGMA) clustering analysis identified three groups for the LP treatment (efficient, intermediate, and inefficient) and three groups for the HP treatment (responsive, moderately responsive, and unresponsive). The results of a principal component analysis and selection index were consistent with the UPGMA analysis, and lines 1, 2, 13, 17, 26, and 31 were classified as PUE.

Treatment of the diabetic foot by offloading: a systematic review.

OBJECTIVE: To compare the literature on the strengths and limitations of different offloading devices in the treatment of diabetic foot ulcers. METHOD: Systematic review of the literature in the following databases: the Cumulative Index to Nursing an Allied Health Literature (CINAHL); Medline; Embase; Cochrane Library and Web of Knowledge. The search strategy used the terms: diabetic foot; orthosis/orthotic devices/orthoses; foot orthosis/foot orthoses; casts/plaster cast/surgical cast; shoes. RESULTS: Our results identified 15 studies, which are included in this review. Healing rates, healing times and reduction in ulcer size were improved with the use of total contact casting, when compared with other offloading devices. The main adverse effects associated with the use of the device were infection, maceration and abrasion. Cost, compliance and quality of life issues were rarely included within the studies. CONCLUSION: Offloading is a key treatment strategy for the management of diabetic foot ulcers and total contact casts were found to be the most effective devices to achieve ulcer healing. However, they are not without complications and their impact on cost, compliance and quality of life is not well understood.

Unexpected co-linearity of Hox gene expression in an aculiferan mollusk.

BACKGROUND: Mollusca is an extremely diverse animal phylum that includes the aculiferans (worm-like aplacophorans and eight-shelled polyplacophorans) and their sister group, the conchiferans, comprising monoplacophorans, bivalves (clams, mussels), gastropods (snails, slugs), scaphopods (tusk shells) and cephalopods (squids, octopuses). Studies on mollusks have revealed an overall number of 11 Hox genes in seven out of eight molluscan "class"-level taxa, but expression data of key developmental regulators such as homeotic genes are only available for three gastropod and two cephalopod species. These show that Hox genes are involved in the formation of specific features including shell, foot, funnel or tentacles and not in antero-posterior body plan patterning as in most other bilaterian animals. The role of Hox genes in non-conchiferan (i.e., aculiferan) mollusks remains entirely unknown. RESULTS: Here we present the first data on the expression of seven Hox genes in apolyplacophoran mollusk, Acanthochitona crinita. In A. crinita the Hox genes Acr-Hox1-5, Hox7 and Post2 are expressed in a co-linear pattern along the antero-posterior axis, but not in molluscan-specific features such as the shell or the foot. The expression pattern is restricted to the post-trochal region and the transcripts are present in ecto-, endo- and mesodermal cell layers. Contrary to the situation in gastropods and cephalopods, we did neither find Hox gene expression in distinct neural subsets of A. crinita, nor in its developing shell plates. CONCLUSIONS: Our analysis and comparison with other lophotrochozoans indicate that the basal role of Hox genes is in antero-posterior axis patterning in mollusks, similar to the vast majority of bilaterian animals, and that this role has been conserved in polyplacophorans, while co-option into patterning of evolutionary novelties emerged either at the base of Conchifera or independently in gastropods and cephalopods. These morphological innovations most likely contributed to the evolutionary success of its representatives, as exemplified by, e.g., the wide ecological range and species richness of gastropods.

Identification, characterisation and molecular modelling of two AP endonucleases from base excision repair pathway in sugarcane provide insights on the early evolution of green plants.

Unlike bacteria and mammals, plant DNA repair pathways are not well characterised, especially in monocots. The understanding of these processes in the plant cell is of major importance, since they may be directly involved in plant acclimation and adaptation to stressful environments. Hence, two sugarcane ESTs were identified as homologues of AP endonuclease from the base-excision repair pathway: ScARP1 and ScARP3. In order to understand their probable function and evolutionary origin, structural and phylogenetic studies were performed using bioinformatics approaches. The two predicted proteins present a considerable amino acid sequence similarity, and molecular modelling procedures indicate that both are functional, since the main structural motifs remain conserved. However, inspection of the sort signal regions on the full-length cDNAs indicated that these proteins have a distinct organelle target. Furthermore, variances in their promoter cis-element motifs were also found. Although the mRNA expression pattern was similar, there were significant differences in their expression levels. Taken together, these data raise the hypothesis that the ScARP is an example of a probable gene duplication event that occurred before monocotyledon/dicotyledon segregation, followed by a sub-functionalisation event in the Poaceae, leading to new intracellular targeting and different expression levels.

Expression of basal lamina components by Schwann cells cultured on poly(lactic acid) (PLLA) and poly(caprolactone) (PCL) membranes.

The present in vitro study investigated the expression of basal lamina components by Schwann cells (SCs) cultivated on PCL and PLLA membranes prepared by solvent evaporation. Cultures of SCs were obtained from sciatic nerves from neonatal Sprague Dawley rats and seeded on 24 well culture plates containing the polymer membranes. The purity of the cultures was evaluated with a Schwann cell marker antibody (anti-S-100). After one week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, laminin I and II. Positive labeling against the studied molecules was observed, indicating that such biomaterials positively stimulate Schwann cell adhesion and proliferation. Overall, the present results provide evidence that membrane-derived biodegradable polymers, particularly those derived from PLLA, are able to provide adequate substrate and stimulate SCs to produce ECM molecules, what may have in turn positive effects in vivo, influencing the peripheral nerve regeneration process.

Rydberg cold collisions dominated by ultralong range potential.

In this work we measure the time evolution of the population resulting from energy-transfer collisions as a function of the energy difference between the entrance and exit collisional channels using a sample of cold Rydberg atoms produced in a rubidium magneto-optical trap. The 34S(1/2) population, produced by collisions between atoms in the 33P(3/2) state, is monitored as a function of time through the pulsed-field ionization technique. The experimental results are compared with a recent published model based on a two-body interaction considering an attractive potential [Phys. Rev. A 65, 023405 (2002)]]; which is calculated according to a recent Letter by Boisseau et al. [Phys. Rev. Lett. 88, 133004 (2002)]]. The agreement is remarkable, which suggests the existence of such ultralong range potential proposed by Boisseau et al.

Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions.

Sugar cane (Saccharum spp.) variety SP 70-1143 was inoculated with Gluconacetobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments. In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted. After 10 d there was extensive anatomical evidence of endophytic colonization by G. diazotrophicus, particularly in lower stems, where high numbers of bacteria were visible within some of the xylem vessels. The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G. diazotrophicus. On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these 'wounds' bacteria were seen colonizing the xylem and intercellular spaces. Bacteria were also occasionally seen entering leaves via damaged stomata, and subsequently colonizing sub-stomatal cavities and intercellular spaces. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion. In experiment 2, stems of 5-week-old greenhouse-grown plants were inoculated by injection with a suspension of G. diazotrophicus containing 10(8) bacteria ml(-1). No hypersensitive response (HR) was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment (7 d). Close to the point of inoculation, G. diazotrophicus cells were observed within the protoxylem and the xylem parenchyma, where they were surrounded by fibrillar material that stained light-green with toluidine blue. In leaf samples taken up to 4 cm from the inoculation points, G. diazotrophicus cells were mainly found within the metaxylem, where they were surrounded by a light green-staining material. The bacteria were growing in relatively low numbers adjacent to the xylem cell walls, and they were separated from the host-derived material by electron-transparent 'haloes' that contained material that reacted with the G. diazotrophicus antibody.

Effect of aggressive prolonged diethylcarbamazine therapy on circulating antigen levels in bancroftian filariasis.

BACKGROUND: Single dose diethylcarbamazine (DEC) as used in control programmes is effectively microfilaricidal for periods of up to a year or more but has incomplete ability to kill Wuchereria bancrofti adult parasites. These regimens can be effective in breaking transmission by suppression of circulating microfilariae available to mosquito vectors. Whether prolonged or aggressive therapy with DEC has a significant effect on adult worms, which may live up to 12 years or more, and is important in the context of the treatment of individual patients, is still incompletely understood. METHODS: In order to investigate the adulticidal effect of aggressive therapy, DEC was given at 6 mg/kg/day for 12-day courses at 0, 6, 12, and 18 months and Og4C3 antigenaemia followed over two years in 38 CAg + Brazilians in a W. bancrofti endemic area. RESULTS: At two year follow-up, the median level of antigenaemia was 21% of the pre-treatment value. 92% of individuals had antigen levels < 50% of pretreatment values, but only 26% had completely cleared antigenaemia. The clearance rate at 24 months was only 12% (3/26) in the asymptomatic CAg + patients but 58% (7/12) in those with clinical manifestations of filariasis. The latter individuals cleared significantly more antigen (median of 0% pretreatment antigenaemia vs. 26%; P=0.02) than asymptomatic but infected individuals. CONCLUSION: Aggressive repeated therapy with DEC alone is ineffective in consistently eradicating adult W. bancrofti, especially in infected but asymptomatic individuals. Prolonged courses of combination therapy with other antifilarial drugs should be investigated for treatment of individual patients with the means to pursue aggressive personal medical care.

Limitations on the clinical utility of the ulnar dorsal cutaneous sensory nerve action potential.

OBJECTIVE: To validate the clinical use of the ulnar dorsal cutaneous (UDCN) sensory nerve action potential (SNAP) in the topographical analysis of ulnar mononeuropathies and in the process of choosing a sensory nerve for biopsy. METHODS: We surveyed the UDCN SNAP electrophysiological characteristics in both hands of 97 normal volunteers aged 10-84 years. The nerve was recorded from the 4th intermetacarpal space with subcutaneous needle electrodes and percutaneous stimulation was carried out at the wrist. RESULTS: In agreement with other studies, the mean conduction velocity was 58. 6+/-6.7 m/s, but the mean and the lower normal value of the amplitude (32 and 14.7 microV) were significantly higher than previous known data. An important finding was that in 21% of our population study, the UDCN SNAP was absent on at least one side, or a significant degree of asymmetry between the left and right sides was present. CONCLUSION: The UDCN SNAP is technically easy to obtain, but the high frequency of asymmetric or absent potentials detected in this study implies that caution should be taken in using this SNAP in clinical practice.

Syringohydromyelia or HTLV-I-associated myelopathy/tropical spastic paraparesis. A diagnostic challenge. Case report.

Human T-cell lymphotropic virus type I (HTLV-I) associated myelopathy/tropical spastic paraparesis (HAM/TSP) is the most common chronic myelopathy in Brazil. We present the case of a 53 year old man that fulfilled the diagnostic criteria for HAM/TSP but had at the magnetic resonance imaging (MRI) of the spinal cord evidences of syringohydromyelia at the C6-C7 and D2-D7 levels along with Chiari type I malformation. The clinical picture was more typical of HAM/TSP than of syringohydromyelia, which was probably asymptomatic. The present case clearly demonstrates that serology and neuroimaging should be always used together. We conclude that, specially in places where HTLV-I is endemic, every patient with a spastic paraparesis, even with a radiological picture suggestive of a structural spinal cord lesion, should have a screening test for HTLV-I. The clinical picture must dictate the final direction of the diagnosis.

[Perianal manifestations in Crohn's disease]. / Manifestações perianais na doença de Crohn.

UNLABELLED: A total of 114 of 195 patients with Crohn's disease had perianal involvement. The average age at the beginning of symptomatology was 30.3 years. The interval between symptoms and diagnosis was 3.1 years. PAC was associated with colonic disease and in these patients, was multiple. PAC preceded intestinal disease in 11 percent, was coincident in 66 percent and appeared later in 23 percent. Sixty one patients (53.5%) were operated on 104 times (1.7 operations per patient). None of these patients developed faecal incontinence. Two patients were treated with hyperbaric oxygenation. The association of perianal disease and extra-intestinal manifestation occurred in 76 patients. There was no association in 38 patients. Forty patients had extra-intestinal manifestation without perianal disease. Twenty two patients had panproctocolectomy because of perianal disease. Twenty one had a stoma, with or without intestinal resection. The stoma improved perianal symptoms, but all remain defunctioned. After mean follow-up of 8.8 years, 45 patients present some kind of perianal complication. CONCLUSION: the surgical treatment of perianal disease well indicated and performed don't result in incontinence; PAC combined with colonic or rectal disease is associated with higher need of performing a proctocolectomy or a defunctioning stoma. Only 22.8 percent presented resolution of perianal disease maintaining anal sphincter function.