Advances in sperm proteomics: best-practise methodology and clinical potential

The recent application of mass spectrometry to the study of the sperm cell has led to an unprecedented capacity for identification of sperm proteins in a variety of species. Knowledge of the proteins that make up the sperm cell represents the first step towards understanding its normal function and the molecular anomalies associated with male infertility. The present review starts with an introduction of the sperm cell biology and is followed by the consideration of the methodological key aspects to be aware of during sample sourcing and preparation, including data interpretation. It then overviews the initiatives developed so far towards the completion of the sperm proteome, with a particular focus in human but with the inclusion of some comments on different model species. Finally, all studies performing differential proteomics in infertile patients are reviewed, pointing to future potential applications.     

Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress

While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.     

Xenon and sevoflurane provide analgesia during labor and fetal brain protection in a perinatal rat model of hypoxia-ischemia

It is not possible to identify all pregnancies at risk of neonatal hypoxic-ischemic encephalopathy (HIE). Many women use some form of analgesia during childbirth and some anesthetic agents have been shown to be neuroprotective when used as analgesics at subanesthetic concentrations. In this study we sought to understand the effects of two anesthetic agents with presumptive analgesic activity and known preconditioning-neuroprotective properties (sevoflurane or xenon), in reducing hypoxia-induced brain damage in a model of intrauterine perinatal asphyxia. The analgesic and neuroprotective effects at subanesthetic levels of sevoflurane (0.35%) or xenon (35%) were tested in a rat model of intrauterine perinatal asphyxia. Analgesic effects were measured by assessing maternal behavior and spinal cord dorsal horn neuronal activation using c-Fos. In separate experiments, intrauterine fetal asphyxia was induced four hours after gas exposure; on post-insult day 3 apoptotic cell death was measured by caspase-3 immunostaining in hippocampal neurons and correlated with the number of viable neurons on postnatal day (PND) 7. A separate cohort of pups was nurtured by a surrogate mother for 50 days when cognitive testing with Morris water maze was performed. Both anesthetic agents provided analgesia as reflected by a reduction in the number of stretching movements and decreased c-Fos expression in the dorsal horn of the spinal cord. Both agents also reduced the number of caspase-3 positive (apoptotic) neurons and increased cell viability in the hippocampus at PND7. These acute histological changes were mirrored by improved cognitive function measured remotely after birth on PND 50 compared to control group. Subanesthetic doses of sevoflurane or xenon provided both analgesia and neuroprotection in this model of intrauterine perinatal asphyxia. These data suggest that anesthetic agents with neuroprotective properties may be effective in preventing HIE and should be tested in clinical trials in the future.     

Floral resources and risk of exposure to pesticides for Melipona quadrifasciata anthidioides Lepeletier 1836 in a Cerrado of São Paulo (Brazil)

Honey and bee bread samples from storage pots of Melipona quadrifasciata anthidioides were collected monthly from April 2015 to May 2016 in the Mogi Guaçu Biological Reserve (22º 10? S, 47º 11? W). The flora in the site is characteristic of the Atlantic Forest with preserved areas of savanna-like vegetation surrounded by commercial forests, orchards and various crops of exotic and native plants. Samples were analysed with the use of melissopalynological methodology and 46 pollen types from 38 genera and 30 families were identified in 25 honey samples. Fabaceae, Asteraceae, Myrtaceae, Sapindaceae showed the greatest pollen richness in honey. Predominant nectariferous pollen types were Anadenanthera, Cordia, Eucalyptus, Mimosa scabrella, Schefflera, Sida, Serjania and Vernonia. Twenty-eight types of pollen from 21 genera and 19 families were identified in 22 bee bread samples. Fabaceae, Asteraceae and Myrtaceae showed the highest pollen richness. Anadenanthera, Cecropia, Eucalyptus, Melastomataceae, Mimosa scabrella, Mimosa verrucosa and Myrcia were the most frequent polliniferous pollen types. Principal component analysis (PCA) demonstrated that honey and pollen samples formed two main groups of similarity, mainly due to Eucalyptus’ nectar and pollen of Melastomataceae, respectively. Melipona quadrifasciata anthidioides collected nectar and pollen from the preserved areas as well as in the secondary and ‘ruderal’ vegetation and in cultivated forests/fields, suggesting their importance as pollinators both of native flora and exotic species. The use of trophic resources of plants grown with pesticides is a concern for the conservation of these species of bee and should be better studied.     

Production and analysis of secondary carotenoids in green algae

The microalgae Neochloris wimmeri, Scenedesmus vacuolatus, Scotiellopsisoocystiformis, Chlorella zofingiensis and Protosiphon botryoides were grown under secondarycarotenoid inductive conditions. The results indicatethat nitrogen deficiency and high light intensity arepotential inducers of astaxanthin formation in thefive microalgae studied. All these microalgaeaccumulate significant quantities of secondarycarotenoids, mainly as astaxanthin esters andcanthaxanthin. They also showed high resistance toenvironmental conditions. All these qualities makethese microalgae good candidates for successfulculture in open ponds.     

<Emphasis Type="Italic">Anemia tomentosa</Emphasis> var. <Emphasis Type="Italic">anthriscifolia</Emphasis> in vitro culture: sporophyte development and volatile compound profile of an aromatic fern

Anemia tomentosa var. anthriscifolia is an aromatic fern with a pleasant woody aroma and antimycobacterial activity. In this paper, we describe for the first time its spore-derived gametophyte development and the effect of indole-3-acetic acid and jasmonic acid on in vitro gametophyte/sporophyte development, as well as volatile compound production. Volatiles were obtained by simultaneous distillation and extraction (SDE) and analyzed by high resolution gas chromatography coupled with mass spectrometry. Spore-derived gametophytes were able to develop into sporophytes independently of the media culture composition, even when no plant growth regulator was added. Fifty different substances were detected in all in vitro A. tomentosa SDE extracts, while 20 were detected in the wild SDE plant extract. Monoterpenes were more prevalent (69.8–89.8%) than sesquiterpenes (9.4–28.7%) in in vitro plants, while sesquiterpenes represent 97.5% of the volatiles produced by the wild-grown plants. The major monoterpene components in in vitro plants were α-pinene (9.3–24.3%), trans-pinocarveol (20.6–27.9%), pinocarvone (15.4–25.1%) and myrtenyl acetate (6.4–12.3%). The triquinane sesquiterpenes silphiperfol-6-ene (0.6–2.9%), α-guaiene (0.5–2.5%), β-barbatene (1.1–3.9%) and 9-epi-presilphiperfolan-1-ol (2.5–5.6%) represent the most abundant sesquiterpenes. The changes in the monoterpene/sesquiterpene rates between micropropagated and wild plants are not related to the presence of JA or IAA in the media culture. Further studies are still needed to obtain a complete understanding of the factors leading to these results, which could be related to differences in the irradiance levels of in vitro plants versus those from a wild environment, as well as the developmental stage of the plants. This is the first report of the use of plant growth regulators on Anemia tomentosa in vitro culture development and their effects on volatile profiles.     

Floral preferences and climate influence in nectar and pollen foraging by Melipona rufiventris Lepeletier (Hymenoptera: Meliponini) in Ubatuba, São Paulo state, Brazil

We describe the environment effects on the amount and quality of resources collected by Melipona rufiventris Lepeletier in the Atlantic Forest at Ubatuba city, S?o Paulo state, Brazil (44o48'W, 23o22'S). Bees carrying pollen and/or nectar were captured at nest entrances during 5 min every hour, from sunrise to sunset, once a month. Pollen loads were counted and saved for acetolysis. Nectar was collected, the volume was determined and the total dissolved solids were determined by refractometer. Air temperature, relative humidity and light intensity were also registered. The number of pollen loads reached its maximum value between 70% and 90% of relative humidity and 18oC and 23oC; for nectar loads this range was broader, 50-90% and 20-30oC. The number of pollen loads increased as relative humidity rose (rs = 0.401; P < 0.01) and high temperatures had a strong negative influence on the number of pollen loads collected (rs = -0.228; P < 0.01). The number of nectar loads positively correlated with temperature (rs = 0.244; P < 0.01) and light intensity (rs = 0.414; P < 0.01). The percentage of total dissolved solids (TDS) on nectar loads positively correlated with temperature and light intensity (rs = 0.361; P < 0.01 and rs = 0.245; P < 0.01), negatively correlated with relative humidity (rs = -0.629; P < 0.01), and it increased along the day. Most nectar loads had TDS between 11% and 30%, with an average of 24.7%. The volume measures did not show any pattern. Important pollen sources were Sapindaceae, Anacardiaceae, Rubiaceae, Arecaceae, Solanaceae and Myrtaceae; nectar sources were Sapindaceae, Fabaceae, Rubiaceae, Arecaceae and Solanaceae.     

The detection and recovery of Erysipelothrix spp. in meat and abattoir samples in Western Australia

AIMS: To investigate the occurrence of Erysipelothrix rhusiopathiae and other Erysipelothrix spp. in abattoir and meat samples in Western Australia. METHODS AND RESULTS: Samples were collected from various parts of pig and sheep carcasses, as well as different sections of slaughtering line, pen soil and effluent. Previously evaluated culture methods were applied for the isolation of Erysipelothrix spp., in conjunction with phenotypic and genotypic detection and identification procedures. Of 109 samples from the two abattoirs, 35 (32.1%) were Erysipelothrix genus-specific PCR-positive. These came from swabs of animal exterior surfaces and joints, slaughtering areas, pig pen soil and abattoir effluent. Four samples (3.7%) from sheep arthritic joints and pig abattoir effluent were also E. rhusiopathiae species-specific PCR-positive. Of 123 carcass washing samples, 12 (9.8%) were genus-specific PCR-positive, and these came from all five kinds of meat samples tested, including beef, lamb, mutton, pork and chicken. Four of them (3.3%) were also species-specific PCR-positive. A total of 25 isolates was recovered from the samples, of which seven were identified as E. rhusiopathiae, seven were consistent with E. tonsillarum, and the remaining 11 were other species of Erysipelothrix. CONCLUSIONS: Erysipelothrix spp. can still be isolated and identified from specimens of animal origin with relative ease, provided that appropriate cultural and molecular procedures are used. Clinical microbiology laboratories may need to improve their diagnostic protocols. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that E. rhusiopathiae and other species of Erysipelothrix continue to colonize and contaminate farmed animals and animal products. Erysipelothrix infection still poses a potential threat to the economy of the farmed animal industry, as well as being a potential human public health hazard.