Prospective external validation of IOTA methods for classifying adnexal masses and retrospective assessment of two-step strategy using benign descriptors and ADNEX: a Portuguese multicenter study.

OBJECTIVE: This study aimed to externally and prospectively validate the International Ovarian Tumor Analysis (IOTA) Simple Rules (SRs), Logistic Regression model 2 (LR2) and Assessment of Different NEoplasias in the adneXa (ADNEX) in a Portuguese population, comparing them with operator subjective assessment (SA), Risk-of-Malignancy Index (RMI), as well as with each other. This study also aimed to retrospectively validate IOTA two-step strategy, using modified benign descriptors (MBDs) followed by the application of ADNEX in cases where MBDs were not applicable (MBDs + ADNEX). METHODS: In this multicenter diagnostic accuracy study, conducted between January 2016 and December 2021, three tertiary referral centers prospectively included consecutive patients with ultrasound diagnosis of at least one adnexal tumor who underwent surgery. All ultrasound assessments were performed by level II or III sonologists with IOTA certification. Patient clinical data and serum cancer antigen (CA125) levels were collected from the hospital databases. Each adnexal mass was classified as benign or malignant using SA, RMI, IOTA SRs, LR2 and ADNEX (with and without CA125). The reference standard was histopathological diagnosis. In the second phase, all adnexal tumors were retrospectively classified using the two-step strategy (MBDs + ADNEX). The sensitivity, specificity, positive (PPV) and negative predictive value (NPV), positive (LR+) and negative likelihood ratio (LR-) as well as overall accuracy were determined for SA, RMI, IOTA SRs, LR2, ADNEX and two-step strategy (MBDs + ADNEX). Receiver-operator characteristic curves were constructed and corresponding areas under the curve (AUC) determined for RMI, LR2 and ADNEX and two-step strategy (MBDs + ADNEX). The ADNEX calibration plots were constructed and estimated by LOESS smoother. RESULTS: Of the 571 included patients, 428 had benign disease, 42 borderline ovarian tumors, 93 primary invasive adnexal cancers and 8 metastatic tumors in adnexa (malignancy prevalence: 25.0%). The operator SA had an overall sensitivity of 97.9% and a specificity of 83.6% for distinguishing between benign and malignant lesions. RMI showed high specificity (95.6%) but very low sensitivity (58.7%), with an AUC of 0.913. The IOTA SRs were applicable in 80.0% of patients, with a sensitivity of 94.8% and a specificity of 98.6%. LR2 revealed a sensitivity of 84.6%, a specificity of 86.9% and an AUC of 0.939, at the malignancy risk cut-off of 10%. At the same cut-off, ADNEX with and without CA125 had a sensitivity of 95.8% and 98.6%, respectively, and a specificity of 82.5% and 79.7%, respectively. The AUC of ADNEX with vs. without CA125 was 0.962 vs. 0.960. The ADNEX model provided heterogeneous results in distinguishing between benign and different subtypes of malignancy, with the highest AUC (0.991) for discriminating benign masses from primary adnexal cancer stage II-IV, and the lowest AUC (0.696) for distinguishing primary adnexal cancer stage I and metastatic lesion in adnexa. The ADNEX calibration plots suggested an underestimation of the predicted risk in relation with the observed proportion of malignancies. The MBDs were applicable in 26.3% of cases (150/571 tumors, none of which were malignant). Similar to the ADNEX model applied in all patients, the two-step strategy using ADNEX in the second step only, with and without CA125, had an AUC of 0.964 and 0.961, respectively. CONCLUSIONS: Our results showed a good to excellent performance of the IOTA methods in the studied Portuguese population, outperforming RMI. ADNEX was superior in accuracy, but interpretation of its ability to distinguish malignant subtypes was fundamentally limited not only by sample size but also by large differences in the prevalence of tumor subtypes. The IOTA MBDs have been shown to be reliable in identifying benign disease. The two-step strategy based on the application of MBDs, followed by the ADNEX model if MBDs are not applicable, has proven to be suitable for daily practice circumventing the need to use electronic support in all patients. This article is protected by copyright. All rights reserved.

Effects of bulk tank milk, waste milk, and pasteurized waste milk on the nutrient utilization, gastrointestinal tract development, and antimicrobial resistance to Escherichia coli in pre-weaned dairy calves.

This study aimed to assess the impact of bulk tank milk (BTM), waste milk (WM), and pasteurized waste milk (PWM) on nutrient digestibility, ruminal and cecal fermentation, gastrointestinal tract (GIT) development, and antimicrobial resistance of fecal Escherichia coli from dairy calves at 2 periods (30 and 60 d of age). Calves were grouped according to body weight, serum protein levels, and breed composition. Three treatments were included: BTM (n = 21), WM from cows under antibiotic treatment (n = 21), and PWM (waste milk submitted to high-temperature, short-time pasteurization; n = 21). A total of 63 calves were used, of which: 18 animals (n = 6 per treatment) evaluated in the period of 4 - 30 d and 45 (n = 15 per treatment) from 4 - 60 d. During the experimental period, a daily intake of 6 L of milk was divided into 2 equal meals, with ad libitum access to water and starter. Milk and feed intakes were recorded daily. Apparent total-tract digestibility and nitrogen balance were conducted from 25 to 29 d of age (n = 6) and from 53 to 57 d of age (n = 15). Animals were euthanized at 30 ± 1 and 60 ± 1 d of age for the assessment of ruminal and cecal fermentation and GIT development. Antimicrobial susceptibility testing was conducted at 1, 30, and 60 d of age (n = 15/treatment). Statistical analysis utilized a linear mixed-effects model for continuous outcomes and generalized linear models for single measurements (R software). Treatments WM and PWM had lower rumen pH, higher ruminal acetate concentration, larger reticulorumen and liver, and a higher prevalence of fecal-resistant E. coli compared with BTM at both 30 and 60 d. Up to 60 d, both BTM and WM treatments exhibited higher digestibility of ether extract and gross energy compared with the PWM, whereas WM and PWM treatments showed increased nitrogen intake and retention compared with the BTM. These findings suggest that pasteurization of waste milk negatively affects nutrient digestibility and calf performance, while also impacting rumen development. Additionally, the use of milk containing antibiotic residue leads to the selection of resistant E. coli in the GIT over time.

Squamous cell carcinoma and basal cell carcinoma of the lips: 25 years of experience in a northeast Brazilian population.

BACKGROUND: The lips are the transition zone between the facial skin and the oral mucosa and are the site of alterations related to a broad spectrum of etiologies. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the most prevalent neoplasms affecting lips. This study evaluated the demographic and clinicopathological features of the SCC and BCC in the lip. MATERIAL AND METHODS: A retrospective cross-sectional descriptive study (1994-2019) was carried out. Demographic and clinicopathologic data were collected from a hospital's dermatological service and an oncologic hospital. The data were submitted to descriptive analysis and Pearson's chi-square and Fisher's exact tests (p ≤ 0.05). RESULTS: 417 medical records were analyzed, of which 323 corresponded to SCC (77.5%) and 94 to BCC (22.5%). SCC showed more frequency in males (58.8%) and BCC in females (54.3%). The lower lip was significantly affected in male patients (p < 0.0001) and by both neoplasms (70.6% and 56.4%, respectively; p = 0.014). SCC and BCC were mainly treated with surgery (88.3% and 93.2%, respectively). Surgical margin was frequently negative in SCC and BCC (87%; 72.3%, respectively), and no recurrence was observed in 79.9% of SCC and 69.1% of BCC cases. CONCLUSIONS: SCC was more frequent in male patients, while BCC showed more frequency in female patients. Both neoplasms mainly affect the lower lip. Understanding the epidemiological profile of these lesions in the lip, as well as their etiology and clinical features, is fundamental for appropriate clinical conduct and the creation and/or amplification of preventive measures.

Increased expression of Filaggrin and Claudin-1 in the ocular surface of patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an itchy, chronic and inflammatory skin condition, with dysfunctional immune response and skin barrier defects. Reduction of filaggrin (FLG) and tight junctions (TJ) proteins, such as claudin-1 (CLDN-1), expression in cutaneous epithelial barrier is remarkable in AD pathogenesis. Ocular involvement occurs in approximately 40% of AD patients leading to changes in the structure of the conjunctiva. OBJECTIVES: We aimed to evaluate the expression of FLG and CLDN-1 in the ocular surface of adults with AD, analysing bulbar conjunctival cells collected by a novel non-invasive cellular imprint. METHODS: Bulbar conjunctival epithelial cells were collected by cellular imprint technique, and FLG and CLDN-1 expression were assessed by immunofluorescence (IF) and real-time polymerase chain reaction (RT-PCR). RESULTS: We detected increased expression of FLG and CLDN-1, as well as their transcript levels in AD patients compared with healthy controls (HC). There was a positive correlation between tear film break-up time (TBUT) and FLG expression. Fluorescein staining was inversely associated with FLG expression. CONCLUSIONS: Our results may reflect a reactive response of the ocular surface to AD-related ocular inflammation and associated dry eye disease. Further investigations focusing on the role of FLG and TJ expression in the ocular surface of AD patients may increment the understanding of the pathophysiology of extracutaneous AD and developing future targeted therapies.

[The effects of quarantine on anxiety and emotional symptoms. Results of an online survey]. / Trastornos de la salud mental en personas sometidas a cuarentena, estudio transversal durante pandemia por COVID-19 en población chilena.

BACKGROUND: Quarantines may exacerbate the presence of emotional symptoms or anxiety. AIM: To explore the relation between time spent in lockdown and development of depressive and anxiety symptoms. MATERIAL AND METHODS: A survey including the GAD anxiety and PHQ-9 depression scores was answered online by 1,488 subjects aged 36 ± 14 years (74% women), invited to participate through social networks. Both scores are validated for the Chilean population. RESULTS: Most responders had a private health insurance system. Sixty seven percent had clinically significant depressive symptoms and 39% had anxiety symptoms. Spending four or more weeks of lockdown (quarantine) was associated with 1.6 times higher risk of developing depressive symptoms and 2.9 times higher risk of developing anxiety symptoms. Difficulties in access to health care increased 3.3 times the risk of developing depression. Suffering a respiratory disease increased 2.39 times the risk of developing anxiety. CONCLUSIONS: There was a direct association between depressive and anxious symptoms, and the time spent of quarantine.

Comparing the efficacy and tolerability of biologic therapies in psoriasis: an updated network meta-analysis.

BACKGROUND: The rapid expansion of psoriasis biologics has led to an urgent need to understand their relative efficacy and tolerability to inform treatment decisions better and, specifically, to inform guideline development. OBJECTIVES: To update a 2017 meta-analysis on the comparative efficacy and tolerability of biologic treatments for psoriasis. METHODS: We searched the MEDLINE, PubMed, Embase and Cochrane databases for randomized controlled trials (RCTs), published up to 7 September 2018, of 11 licensed, NICE-approved biologics targeting tumour necrosis factor (adalimumab, etanercept, infliximab, certolizumab pegol), interleukin (IL)-12/IL-23p40 (ustekinumab), IL-17A (secukinumab, ixekizumab), IL-17RA (brodalumab) and IL-23p19 (guselkumab, tildrakizumab, risankizumab). A frequentist network meta-analysis ascertained direct or indirect evidence comparing biologics with one another, methotrexate or placebo. This was combined with hierarchical cluster analyses to consider efficacy (≥ 90% improvement in Psoriasis Area and Severity Index (PASI 90) or Physician's Global Assessment 0 or 1; PASI 75; Dermatology Life Quality Index improvement) and tolerability (drug withdrawal due to adverse events) outcomes at 10-16 weeks, followed by assessments of study quality, heterogeneity and inconsistency. RESULTS: We identified 62 RCTs presenting data on direct comparisons (31 899 participants). All biologics were efficacious compared with placebo or methotrexate at 10-16 weeks. Hierarchical cluster analyses revealed that adalimumab, brodalumab, certolizumab pegol, guselkumab, risankizumab, secukinumab, tildrakizumab and ustekinumab were comparable with respect to high short-term efficacy and tolerability. Infliximab and ixekizumab clustered together, with high short-term efficacy but relatively lower tolerability than the other agents, although the number of drug withdrawal events across the network was low, so these findings should be treated with caution. CONCLUSIONS: Using our methodology we found that most biologics cluster together with respect to short-term efficacy and tolerability, and we did not identify any single agent as 'best'. These data need to be interpreted in the context of longer-term efficacy, effectiveness data, safety, posology and drug acquisition costs when making treatment decisions.

Comparison of antibody repertories against Staphylococcus aureus in healthy and infected dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine.

Staphylococcus aureus is one of the pathogens most frequently isolated from cases of mastitis worldwide. To decrease the effect of S. aureus mastitis in dairy farming, alternative strategies for controlling mastitis are needed that depend on a better knowledge of cow-to-cow variations in S. aureus antibody production. The present study sought to explore the diversity of S. aureus antibodies produced by dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine. We obtained protein extracts from S. aureus isolates derived from persistent subclinical mastitis. Proteins were fractionated using 2-dimensional gel electrophoresis and Western blotting. Then, Western blotting membranes were exposed to sera from 24 dairy cows that had been divided into the following groups: vaccinated dairy cows that were infected with S. aureus, further subdivided according to whether they (a) remained infected by S. aureus or (b) recovered from the intramammary infection; unvaccinated dairy cows infected with S. aureus; and vaccinated healthy dairy cows with no history of S. aureus mastitis. Proteins found to be reactive by Western blot were identified by mass spectrometry (MALDI/TOF-TOF). Our most important finding was that F0F1 ATP synthase subunit α, succinyl-diaminopimelate desuccinylase, and cysteinyl-tRNA synthetase were potential candidate proteins for the prevention of S. aureus mastitis. This study strengthens the notion that variations among animals should not be ignored and shows that the heterogeneity of antibody production against anti-staphylococcal antigens in animals may enable the identification of new immunotherapy targets.

Blood-brain barrier transport and neuroprotective potential of blackberry-digested polyphenols: an in vitro study.

PURPOSE: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood-brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. METHODS: BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC-MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. RESULTS: Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. CONCLUSIONS: BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours).

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post-mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer-assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3'3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.

A Commensal Bifidobacterium longum Strain Prevents Gluten-Related Immunopathology in Mice through Expression of a Serine Protease Inhibitor.

Microbiota-modulating strategies, including probiotic administration, have been tested for the treatment of chronic gastrointestinal diseases despite limited information regarding their mechanisms of action. We previously demonstrated that patients with active celiac disease have decreased duodenal expression of elafin, a human serine protease inhibitor, and supplementation of elafin by a recombinant Lactococcus lactis strain prevents gliadin-induced immunopathology in the NOD/DQ8 mouse model of gluten sensitivity. The commensal probiotic strain Bifidobacterium longum NCC2705 produces a serine protease inhibitor (Srp) that exhibits immune-modulating properties. Here, we demonstrate that B. longum NCC2705, but not a srp knockout mutant, attenuates gliadin-induced immunopathology and impacts intestinal microbial composition in NOD/DQ8 mice. Our results highlight the beneficial effects of a serine protease inhibitor produced by commensal B. longum strains.IMPORTANCE Probiotic therapies have been widely used to treat gastrointestinal disorders with variable success and poor mechanistic insight. Delivery of specific anti-inflammatory molecules has been limited to the use of genetically modified organisms, which has raised some public and regulatory concerns. By examining a specific microbial product naturally expressed by a commensal bacterial strain, we provide insight into a mechanistic basis for the use of B. longum NCC2705 to help treat gluten-related disorders.

How does temperature influences the development of lactococcosis? Transcriptomic and immunoproteomic in vitro approaches.

Lactococcus garvieae is the aetiological agent of lactococcosis, a haemorrhagic septicaemia that affects marine and freshwater fish, with special incidence and economic relevance in farmed rainbow trout. Water temperature is one of the most important predisposing factors in the development of lactococcosis outbreaks. Lactococcosis in trout usually occur when water temperatures rise to about 18 °C, while fish carriers remain asymptomatic at temperatures below 13 °C. The aim of this work was to analyse the differences in the complete transcriptome response of L. garvieae grown at 18 °C and at 13 °C and to identify the immunogenic proteins expressed by this bacterium at 18 °C. Our results show that water temperature influences the expression of L. garvieae genes involved in the lysis of part of the bacterial cell population and in the cold response bacterial adaptation. Moreover, the surface immunogenic protein profile at 18 °C suggests an important role of the lysozyme-like enzyme, WxL surface proteins and some putative moonlighting proteins (proteins with more than one function, usually associated with different cellular locations) as virulence factors in L. garvieae. The results of this study could provide insights into the understanding of the virulence mechanisms of L. garvieae in fish.

An optimized protocol for DNA extraction in plants with a high content of secondary metabolites, based on leaves of Mimosa tenuiflora (Willd.) Poir. (Leguminosae).

Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.

Uterine haemodynamic, vascularization and blood pressure changes along the oestrous cycle in bitches.

Characterization of the bitch's reproductive physiology is of utmost importance for the development of new reproductive techniques and the diagnosis of reproductive diseases. In this respect, uterine B-mode ultrasonography has been employed in several studies; however, the focus on haemodynamic changes along the oestrous cycle is yet to be described. Thus, the aim of this study was to characterize haemodynamic changes (uterine vascularization, systemic arterial blood pressure and heart rate) throughout the oestrous cycle in bitches. For this purpose, ten Golden Retriever bitches were evaluated during an entire oestrous cycle, twice during each stage of the cycle. Uterine artery blood flow, velocity wave forms, haemodynamic parameters and vascularization were analysed by Doppler ultrasonography. Furthermore, uterine diameter, arterial blood pressure and heart rate were measured. Uterine artery pulsatility index at early prooestrus was significantly lower in comparison with early oestrus, mid- and late anoestrus. Uterine artery resistance index was higher at early oestrus when compared to late oestrus and uterine diameter was significantly higher during late prooestrus. Furthermore, mean arterial blood pressure was lower and heart rate was higher during late prooestrus in comparison with the other oestrous cycle stages. In conclusion, haemodynamic changes in the uterine artery, uterine diameter, systemic blood pressure and heart rate occur during the canine oestrous cycle. Specifically, there is an increase in uterine artery perfusion, uterine diameter and mean arterial blood pressure during prooestrus, while uterine blood flow diminishes during oestrus and anoestrus.

The influence of benign prostatic hyperplasia on sperm morphological features and sperm DNA integrity in dogs.

Benign prostatic hyperplasia (BPH) has a high incidence in older intact dogs. Due to the increased prostatic oxidative stress and hormonal imbalance of BPH, sperm damage can arise, such as sperm morphological alterations and DNA fragmentation. This study aimed to compare the reproductive potential of healthy dogs and those affected by benign prostatic hyperplasia. Ten dogs were assigned to two experimental groups: dogs without BPH (control; n = 5) and dogs diagnosed with BPH (n = 5), based on clinical signs and ultrasonographic findings. Three semen collections were performed from each dog within one month and analysed using computer-assisted sperm analysis (CASA) and functional tests. Control group showed higher percentage of sperm DNA integrity (95 ± 1.8%) compared to the BPH group (79.2 ± 6.4%). On the other hand, the percentage of minor sperm defects, amplitude of lateral sperm head displacement of the spermatozoa and medium sperm mitochondrial activity were higher in the BPH group. In conclusion, BPH decreases sperm DNA integrity, increases mitochondrial activity, as well as modifies sperm movement pattern. Therefore, a careful sperm analysis of aged dogs with BPH is required before a reproductive programme can be established for such patients.

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2-deoxy-d-glucose, DOG). Furthermore, treatment with DOG also led to a dose-dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility.

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

SHORT-COMMUNICATION Evaluation of perfused bovine udder for gene expression studies in dairy cows.

Intramammary infections are one of the main causes of productivity loss in dairy cows. To better understand the immune system response and to avoid the use of live animals, we validated the use of isolated bovine udder as an ex situ model. Six mammary glands were collected from cows ready for culling. Three udders were perfused with Tyrode's solution and three were not-perfused. During six hours, we collected perfusate samples for biochemical analysis. We also collected alveolar and teat canal tissue to evaluate gene expression. The biochemical parameters indicated that the perfused udders remained viable for the entire period of the experiment. A real-time polymerase chain reaction showed an increase in 18S rRNA gene expression in the alveolar tissue at 3 and 4 h after perfusion. There was also an increase in the Ubiquitin gene in the teat canal from not-perfused udders at 1, 3, and 4 h after slaughter. In general, gene expression was stable during the experiment. Our results indicated that the isolated perfused bovine udder model is appropriate for genetic studies, opening a new perspective in animal experimentation methods.

Cellular response of the blood-brain barrier to injury: Potential biomarkers and therapeutic targets for brain regeneration.

Endothelial cells are the main component of the blood-brain barrier (BBB), a vital structure for maintaining brain homeostasis that is seriously disrupted in various neurological pathologies. Therefore, vascular-targeted therapies may bring advantages for the prevention and treatment of brain disorders. In this sense, novel methods to identify and evaluate endothelial damage have been developed and include the detection of circulating endothelial cells, endothelial progenitor cells, endothelial microparticles and exosomes. These cells and cellular structures have been documented in numerous diseases, and increasingly in neurodegenerative disorders, which have led many to assume that they can either be possible biomarkers or tools of repair. Therefore, the purpose of this review is to discuss available data on BBB endothelial damage occurring in two pathologies of the central nervous system, Alzheimer's disease and stroke, which exemplify conditions where chronic and acute vascular damage occur, respectively. The ultimate goal is to identify useful biomarkers and/or therapeutic tools in the healthy and diseased brain that can be used for the treatment of neurodegenerative diseases where BBB permeability and integrity are impaired.