Prostaglandin-related immune suppression in cattle.

Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.

The interplay between autophagy and apoptosis mediates toxicity triggered by synthetic cathinones in human kidney cells.

Synthetic cathinones abuse remains a serious public health problem. Kidney injury has been reported in intoxications associated with synthetic cathinones, but the molecular mechanisms involved have not been explored yet. In this study, the potential in vitro nephrotoxic effects of four commonly abused cathinone derivatives, namely pentedrone, 3,4-dimethylmethcatinone (3,4-DMMC), methylone and 3,4-methylenedioxypyrovalerone (MDPV), were assessed in the human kidney HK-2 cell line. All four derivatives elicited cell death in a concentration- and time-dependent manner, in the following order of potency: 3,4-DMMC >> MDPV > methylone ≈ pentedrone. 3,4-DMMC and methylone were selected to further elucidate the mechanisms behind synthetic cathinones-induced cell death. Both drugs elicited apoptotic cell death and prompted the formation of acidic vesicular organelles and autophagosomes in HK-2 cells. Moreover, the autophagy inhibitor 3-methyladenine significantly potentiated cell death, indicating that autophagy may serve as a cell survival mechanism that protects renal cells against synthetic cathinones toxicity. Both drugs triggered a rise in reactive oxygen and nitrogen species formation, which was completely prevented by antioxidant treatment with N­acetyl­L­cysteine or ascorbic acid. Importantly, these antioxidant agents significantly aggravated renal cell death induced by cathinone derivatives, most likely due to their autophagy-blocking properties. Taken together, our results support an intricate control of cell survival/death modulated by oxidative stress, apoptosis and autophagy in synthetic cathinones-induced renal injury.

Tracheostomy prevalence at Skilled Nursing Facilities.

The incidence of chronically ill subjects with prolonged mechanical ventilation has significantly increased over the last decade. Many patients get discharge to Skilled Nursing Facilities with an artificial airway, which do not have the means to properly progress on weaning. In Portugal this prevalence is unknown. Our aim was to establish the prevalence of tracheostomized patients at SNF in the North of Portugal, characterizing these units and its population, in a cross-sectional study, through an online questionnaire answered on the same day. Of the 75 SNF, 30 answered: 13 long-term, 2 medium-term, 2 short-term, 12 had beds of both medium and long-term and 1 had the three typologies. 33 had tracheostomy ventilation (prevalence 3.36%), all admitted at long-term units, the majority transferred from previous hospital admission (n=27, 90%). Only one was under mechanical ventilation. The most frequent reason for tracheostomy placement was acute respiratory failure (n=10, 33.3%). The most commonly presented cannula was the fenestrated non-cuffed (n=17, 59%). Only 4 were performing occlusion training, 21 needed frequent secretion suctioning and 1 used the mechanical in-exsufflation. Regarding motor function, 16 (53.3%) were unable to achieve sitting balance and 20 (66.7%) had no orthostatic balance or walking ability. 14 (46.7%) had percutaneous endoscopic gastrostomy. Although low response rate may induce some bias, this study revealed a significant prevalence of tracheostomized patients at SNF. These facilities do not have the resources to safely and effectively progress on ventilatory weaning. It is essential to establish new referral criteria and create specialized weaning units.

Antibacterial effect and biocompatibility of a novel nanostructured ZnO-coated gutta-percha cone for improved endodontic treatment.

This work explored a novel approach to enhance the antibacterial activity of commercial Gutta-percha (GP) cones, the most commonly used core filling materials used in endodontic treatment. The reported procedure involved an argon (Ar) plasma treatment (PT) of the GP cone surface, followed by the deposition of a ZnO thin film by magnetron sputtering. The resulting surfaces were evaluated for surface topography, antibacterial activity against Enterococcus faecalis and Staphylococcus aureus, and cytocompatibility with human osteoblastic cells. GP cones treated with NaOCl, a routine chair-side protocol, were also tested as reference. The deposition of a ZnO film on pristine GP cones increased its antibacterial activity. Cones pre-treated with Ar-plasma (PT) and coated with the ZnO thin film presented significantly higher antibacterial activity than that observed on the pristine and, also, compared to the ZnO coated cones. The higher antibacterial activity of PT + ZnO cones appears related to the major effects induced by the PT pre-treatment on the cone surface endowing the deposited ZnO film with a homogeneous nanostructured topography that greatly improved surface reactivity. The modified GP cones maintained an appropriate cytocompatibility with human cells. This novel approach provides ready-to-use cones with enhanced antibacterial activity, improving a strict asepsis protocol during endodontic treatment and preventing secondary endodontic infections.

Generation of patient-specific induced pluripotent stem cell lines from one patient with Jervell and Lange-Nielsen syndrome, one with type 1 long QT syndrome and two healthy relatives.

Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.

Measurements of a 'normal vulva' in women aged 15-84: a cross-sectional prospective single-centre study.

OBJECTIVE: Accurate and detailed description of a 'normal vulva' is rare, even though a few studies have dealt with the topic of normal measurements of parts of the external female genitalia. This leads to a situation with a wide range of existing diagnoses concerning 'normal' vulvar morphology. DESIGN: Cross-sectional prospective single-centre study. SETTING: From August 2015 to April 2017, we included 657 women in our gynaecological and uro-gynaecological outpatient clinic of the Cantonal Hospital Lucerne. POPULATION OR SAMPLE: We recruited white women aged 15-84 years. METHODS: Standardised defined measurements were taken of the clitoral gland, distance from the base of the gland to the urethral orifice, length of introitus, length of perineum, length of labia majora, and length and width of labia minora. Furthermore, we recorded baseline characteristics. MAIN OUTCOME MEASURES: The length of labia minora (r = -0.364, P < 0.001, n = 657) as well as the length of the perineum (r = -0.095, P = 0.014, n = 657) are inversely correlated with age. A positive correlation between body mass index and the length of the labia majora (r = 0.150, P < 0.001, n = 657) and the length of the introitus (r = 0.097, P = 0.014, n = 657) was found. RESULTS: We provide detailed data on age-related dimensions and description of the external female genitalia in a homogeneous group of white women. CONCLUSION: With our data, we present a baseline for the appearance of a normal white vulva, which could be used to establish standards for indications for gynaecological cosmetic surgery. TWEETABLE ABSTRACT: This study presents data on the standard dimensions of the external female genitalia and is, to our knowledge, the biggest cohort presented on this topic by now.

A soluble inorganic pyrophosphatase from the cattle tick Rhipicephalus microplus capable of hydrolysing polyphosphates.

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been investigated mainly in mammalian cells, and only a few studies have addressed arthropods. Pyrophosphatases have been shown to regulate polyphosphate metabolism. However, these studies were restricted to trypanosomatids. Here we focus on the tick Rhipicephalus microplus, a haematophagous ectoparasite that is highly harmful to cattle. We produced a recombinant R. microplus pyrophosphatase (rRmPPase) with the aim of investigating its kinetic parameters using polyphosphates as substrate. Molecular docking assays of RmPPase with polyphosphates were also carried out. The kinetic and Hill coefficient parameters indicated that rRmPPase has a greater affinity, higher catalytic efficiency and increased cooperativity for sodium phosphate glass type 15 (polyP15 ) than for sodium tripolyphosphate (polyP3 ). Through molecular docking, we found that polyP3 binds close to the Mg2+ atoms in the catalytic region of the protein, participating in their coordination network, whereas polyP15 interactions involve negatively charged phosphate groups and basic amino acid residues, such as Lys56, Arg58 and Lys193; polyP15 has a more favourable theoretical binding affinity than polyP3 , thus supporting the kinetic data. This study shows, for the first time in arthropods, a pyrophosphatase with polyphosphatase activity, suggesting its participation in polyphosphate metabolism.

Peptidase inhibitors in tick physiology.

Peptidase inhibitors regulate a wide range of physiological processes involved in the interaction between hematophagous parasites and their hosts, including tissue remodeling, the immune response and blood coagulation. In tick physiology, peptidase inhibitors have a crucial role in adaptation to improve parasitism mechanisms, facilitating blood feeding by interfering with defense-related host peptidases. Recently, a larger number of studies on this topic led to the description of several new tick inhibitors displaying interesting novel features, for example a role in pathogen transmission to the host. A comprehensive review discussing these emerging concepts can therefore shed light on peptidase inhibitor functions, their relevance to tick physiology and their potential applications. Here, we summarize and examine the general characteristics, functional diversity and action of tick peptidase inhibitors with known physiological roles in the tick-host-pathogen interaction.

Characterization of a glycine-rich protein from Rhipicephalus microplus: tissue expression, gene silencing and immune recognition.

Salivary molecules, as glycine-rich proteins (GRPs), are essential to tick attachment and feeding on the host and are suggested to be involved in the host's immune system evasion, therefore representing natural candidates in the search for protective vaccine antigens. This work shows the molecular characterization of a GRP from Rhipicephalus microplus (RmGRP). The cDNA and putative amino acid sequences were analysed, as well as the transcription level in tick tissues/developmental stages, showing the highest levels of gene expression in 1-day-old larvae and salivary glands of fully engorged females. RmGRP gene silencing resulted in a lower hatching rate of larvae from treated females. In addition, recombinant RmGRP (rRmGRP) was recognized by sera from naturally and experimentally infested bovines, displaying considerable differences among the individuals tested. rRmGRP was recognized by anti-saliva and anti-salivary glands sera, while anti-rRmGRP serum recognized RmGRP in saliva and salivary glands, indicating its secretion into the host. The data collected indicate that RmGRP may present roles other than in the tick-host relationship, especially in embryo development. In addition, the high expression in adult females, antigenicity and presence of shared characteristics with other tick protective GRPs turns RmGRP a potential candidate to compose an anti-tick vaccine cocktail.

Colostrum yield and litter performance in multiparous sows subjected to farrowing induction.

The consumption of colostrum at a low level can compromise the survival and growth of piglets. Therefore, this study aimed to evaluate the effect of farrowing induction on colostrum yield, IgG concentration and the survival and performance of piglets until the weaning. Sows of parity 3 to 7 were assigned into two groups: Control (n = 48), sows with spontaneous farrowing; and induction (n = 48), sows induced to farrow on day 114 of gestation with a PGF2 analogue. Colostrum and blood samples were collected from the sows, at farrowing and 24 hr later. Blood samples from the piglets were collected at 24 hr after birth. The performance of the piglets was evaluated in a subsample of 28 litters from each group. All piglets were weighed at 7, 14 and 20 days of age. The farrowing length, the number of piglets born alive, stillborn piglets, weight at birth, litter weight at birth and colostrum yield were not significantly affected (p > .05) by farrowing induction. There was no difference between the groups (p > .05) in the percentage of sows with obstetric interventions. Serum IgG concentration, in both sows and piglets, and colostrum IgG concentration were similar between the groups (p > .05). Furthermore, survival rate, piglet weight and litter weight at 7, 14 and 20 days of age were also similar between the groups (p > .05). Therefore, it can be concluded that the farrowing induction performed on day 114 of gestation does not affect the colostrum yield, the IgG concentration in colostrum and serum of piglets, and the litter performance until the weaning.

Notification of Huntington's disease as primary cause of death in Brazil from 1984 to 2008.

The aim of this article was to conduct a retrospective observational study on reported deaths due to Huntington's disease (HD) in Brazil in the past 25 years (from 1984 to 2008). Data were obtained from the Brazilian Mortality Information System (SIM/DATASUS), the official system of Brazilian Mortality Database. The data obtained included information regarding the gender of the deceased and the number of death notifications, which we stratified by demographic regions and states. HD mortality per 100,000 was calculated and plotted in a graph. Linear regression was calculated using ordinary least square technique. We observed that the mortality due to HD recorded by SIM/DATASUS from 1984 to 2008 had increased at much higher rates than the population in the same period. Also, some Brazilian regions still show very low rates of HD mortality compared to the national average of deaths due to HD. These findings suggest that HD mortality has been underestimated. Ignorance about the disease as well as the fact that death from HD can occur as a consequence of heart disease, pneumonia, or suicide can strongly contribute to the misguided notification of HD as the cause of death in the official reports.

Effect of birth weight and colostrum intake on mortality and performance of piglets after cross-fostering in sows of different parities.

The objective of this study was to evaluate the effect of colostrum intake on mortality and growth performance of piglets until 42 days of age, taking into account the birth weight, and parity order of sows. Colostrum intake from birth to 24h after birth was estimated in 300 piglets each from primiparous and multiparous sows. The piglets were cross-fostered in 25 primiparous and 25 multiparous sows at 25.9±0.09 h after farrowing. The concentration of serum immunoglobulin G (IgG) was determined in the sows after the end of farrowing and in the piglets before cross-fostering (24h after farrowing), at 10 and 20 days of age. For high birth weight piglets (HBW - >1.3-1.7 kg) the probability of death was low regardless of their colostrum intake. Intermediate birth weight (IBW - >1.2-1.3 kg) and low birth weight (LBW - 1.1-1.2 kg) piglets had the same probability of death compared to HBW piglets, when colostrum intake reached 200 and 250 g, respectively. The probability of low performance (<9.5 kg) was lower in HBW than in LBW and IBW piglets, regardless of colostrum intake. HBW piglets had higher weight at 14, 20, 28 and 42 days of age (P<0.05) than LBW piglets, and higher weight than IBW piglets at 28 and 42 days. Colostrum intake >250 g increased (P<0.05) the weight of piglets at 28 and 42 days of age, regardless of their birth weight. Piglets from primiparous biological dams consumed less colostrum (P<0.003) than piglets from multiparous dams, but their serum IgG concentrations at 24h after birth and their performance were similar (P>0.05). Piglets suckled by primiparous foster sows showed lower weight (P<0.05) at 20, 28 and 42 days than piglets suckled by multiparous sows. Piglets that died before 42 days of age had lower (P<0.05) birth weight, colostrum intake and serum IgG at 24 h after birth compared to surviving piglets, and lower values were also observed in piglets with low performance compared to high (>9.5 kg) performance piglets. There were no differences in serum IgG concentrations at 10 and 20 days of age (P>0.05) between high and low performance piglets. In conclusion, both colostrum intake and birth weight are important for survival and growth of suckling piglets. LBW and IBW piglets are more dependent on colostrum intake than HBW piglets to assure higher survival and better development up to 42 days of age. Piglets suckled by multiparous sows have better development than those suckled by primiparous sows.

Antibacterial, physicochemical and mechanical properties of endodontic sealers containing quaternary ammonium polyethylenimine nanoparticles.

AIM: To evaluate the antibacterial, physicochemical and mechanical properties of two endodontic sealers incorporating quaternary ammonium polyethylenimine (QPEI) nanoparticles at concentrations of 1% and 2% (w/w). METHODOLOGY: The sealers tested were AH Plus and Pulp Canal Sealer EWT in the commercial unmodified form or containing 1% or 2% QPEI. Antibacterial activity was evaluated using a direct contact test (DCT) against Enterococcus faecalis ATCC 29212 and two endodontic isolates (RW35 and RN44). Sealers freshly mixed or set for 7 days were exposed to bacterial suspensions for 10, 30 and 60 min. Setting time, flow test, solubility, apparent porosity, dimensional change following setting, wettability, zeta potential and compressive strength were assessed according to the International Standard Organization 6876:2001 (ISO 6876). RESULTS: DCT results revealed that both freshly prepared sealers had antibacterial effects unaffected by QPEI incorporation. Both unmodified sealers had lost much of their antibacterial effects after 7 days. However, for Pulp Canal Sealer EWT incorporated with 1% and 2% QPEI nanoparticles, the antibacterial effects against all test E. faecalis strains within 30 and 60 min of contact were significantly greater than the unmodified formula. Addition of QPEI resulted in no significant increase in the antibacterial effects of AH Plus after ageing. As for physicochemical and mechanical tests, setting time, wettability and zeta potential were influenced by the presence of QPEI nanoparticles. CONCLUSION: Incorporation of QPEI nanoparticles can improve the long-term antibacterial activity of Pulp Canal Sealer EWT without relevant changes in physicochemical and mechanical properties.

Efficacy of Er,Cr:YSGG laser with endodontical radial firing tips on the outcome of endodontic treatment: blind randomized controlled clinical trial with six-month evaluation.

Clinical reports stating the efficacy of novel root canal disinfection protocols are an important focus in endodontic research. This blind randomized clinical trial assessed the clinical efficacy of the erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser radial firing tips (RFT) versus the concomitant use of 3 % sodium hypochlorite and interim calcium hydroxide paste in necrotic teeth with chronic apical periodontitis. We hypothesized to find similar or improved bone healing in the laser-assisted endodontic treatment. Thirty-six anterior and premolar teeth were randomly assigned. In group 1, teeth were prepared with 3 % sodium hypochlorite for irrigation and calcium hydroxide as inter-appointment dressing; in group 2, teeth were prepared with saline solution and irradiated with Er,Cr:YSGG laser using RFT2 (140 µs, 37.5 mJ, 20 Hz) and RFT3 (140 µs, 62.5 mJ, 20 Hz) in the first and second appointment, respectively, four times each, moving at 2 mm s(-1) from apical to coronal. The primary outcome measure was changed in apical bone density at 6 months, using the periapical index (PAI) for blind radiographic evaluation. Twenty-nine patients were examined and subjected to statistical analysis, 12 in group 1 and 17 in group 2. There was one treatment failure in group 1. Both groups gave similar outcomes exhibiting statistically significant decreases in PAI scores.

Systemic alterations of bovine hemostasis due to Rhipicephalus (Boophilus) microplus infestation.

The tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that causes considerable economic losses to cattle breeding. Although R. microplus saliva contains several molecules that interfere with the blood coagulation process, so far the systemic alterations in the host hemostatic system have not been described. This study aims to determine if R. microplus infestation induces any disturbance to the host's hemostatic system. To address these questions, six calves were experimentally infested with 20,000 R. microplus larvae and their blood was collected before and 7, 14 and 21 days post-infestation. Collagen and ADP-induced platelet aggregation as well as coagulation (activated partial thromboplastin time and prothrombin time) decreased in infested bovines. Platelet blood count and fibrinogen increased during the course of infestation, probably as a compensatory response. These alterations may play a role in host health status, and show that the host cannot fully counteract the tick anti-hemostatic mechanisms.

Comparative IgG recognition of tick extracts by sera of experimentally infested bovines.

Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to investigate the pattern of antibody responses of six bovines infested twelve times with Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae) (six heavy infestations followed by six light infestations) against salivary gland, gut and larvae extracts. During heavy infestations, bovine IgG levels were shown to be higher, and a decrease in the number and weight of ticks that completed the parasitic cycle was observed. The pattern changed starting from the seventh infestation, showing a decrease in IgG levels. An initial increase followed by a significant decrease in the proportion of ticks that completed the parasitic cycle was also observed from the seventh infestation. The number of molecules recognized by Western blot was higher from sera collected following heavy infestations than after light infestations, although a great variation in the profiles detected could be seen when the bovines were compared. These results indicate that IgG responses to different tick antigens may not be generally associated with bovine resistance, and that infestation levels modulate the magnitude of humoral responses and possibly the immune mechanisms in the natural acquisition of tick resistance.

Relationship between glutathione S-transferase, catalase, oxygen consumption, lipid peroxidation and oxidative stress in eggs and larvae of Boophilus microplus (Acarina: Ixodidae).

Glutathione S-transferases (GSTs) are enzymes that act in excretion of physiologic and xenobiotic substances, protecting cells against chemical toxicity and stress. In this work, we characterized the enzymatic activity of GST in eggs and larvae of cattle tick Boophilus microplus, on different days after oviposition and eclosion. The results showed that the GST activity varied depending on the time elapsed after oviposition and eclosion. Molecules involved in mechanism of protection from oxidative stress are correlated with the increase in GST activity. The oxygen consumption kinetics showed a positive correlation with the increase in GST activity during embryogenesis. A high content of thiobarbituric acid reactive substances were observed in egg and larva extracts, indicating that ticks face high oxidative stress during embryogenesis and aging. In eggs and larvae, GST activity can be correlated to kinetic parameters of oxidative stress such as catalase and glutathione. In addition, GST activity showed strong positive correlation with lipid peroxidation, an indication that it plays a role in oxidant defences in eggs.

Cell death during preoviposition period in Boophilus microplus tick.

Programmed cell death (PCD) is present during the development of multicellular organisms and occurs from embryogenesis to death. In females of Boophilus microplus, the mass of several organs is reduced after the detachment from the host. In order to better characterize the cell death process that eliminates unnecessary tissues, the degeneration of salivary glands, ovaries and synganglia was investigated using DNA fragmentation in agarose gel, comet and TUNEL assays, and apoptosis activation pathway by the caspase assay. DNA fragmentation and enzymatic activity of caspase-3 were observed in salivary glands and ovaries at 48 and 72h after tick removal from the host; in synganglia these parameters were maintained at low levels upon 48h. The results obtained suggest that there is a refined control of tissue maintenance through apoptosis.