Confirmatory assays for transient changes of omics in soil invertebrates - Copper materials in a multigenerational exposure.

Environmental risk assessment (ERA) based on effects caused by chronic and longer term exposure is highly relevant. Further, if mechanistic based approaches (e.g. omics) can be included, beyond apical endpoints (e.g. reproduction), the prediction of effects increases. For Cu NMs (and CuCl2) this has been studied in detail, covering multi-omics and apical effects using the soil standard species Enchytraeus crypticus. The intermediate level effects like cell/tissue and organ alterations represent a missing link. In the present study we aimed to: 1) perform long term exposure to Cu materials (full life cycle and multigeneration, 46 and 224 days) to collect samples; 2) perform histology and immunohistochemistry on collected samples at 12 time points and 17 treatments; 3) integrate all levels of biological organization onto an adverse outcome pathway (AOP) framework. CuO NMs and CuCl2 caused both similar and different stress response, either at molecular initiating events (MIE) or key events (KEs) of higher level of biological organization. Cell/Tissue and organ level, post-transcriptional and transcriptional mechanisms, through histone modifications and microRNA related protein, were similarly affected. While both Cu forms affected the Notch signalling pathway, CuCl2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl2 at the MIE.

Comparative overview of the effects of aerobic and resistance exercise on anxiety-like behavior, cognitive flexibility, and hippocampal synaptic plasticity parameters in healthy rats.

Clinical studies show that physical exercise has anxiolytic and pro-cognitive properties for both healthy individuals and psychiatric patients. Most of these data refer to the effects of aerobic exercise. However, other modalities such as resistance exercise deserve more attention because they may also modulate brain function. This study aimed to compare the effects of an aerobic exercise protocol on a treadmill and a resistance exercise protocol on a ladder apparatus on anxiety-like behavior, cognitive flexibility, and neuroplasticity parameters in healthy animals. Adult male Wistar rats were divided into three groups: sedentary control, aerobic training, and resistance training. Subsequently, they were evaluated in the elevated plus-maze (EPM), light-dark box, and modified hole board (mHB) tests. The expressions of synaptophysin and postsynaptic plasticity protein 95 in the dorsal and ventral hippocampus were analyzed by immunofluorescence. The results demonstrated an anxiolytic effect promoted by exercise in the EPM, particularly in the animals submitted to aerobic training, and a mild pro-learning effect of both exercise modalities was observed in the mHB test. All groups showed similar outcomes in the other evaluations. Therefore, the exercise modalities investigated in the present study did not provide considerable modifications to such aspects of the emotional/cognitive functions and neuroplasticity under physiological contexts. Perhaps the two types of exercise acted in neurobiological pathways not analyzed in this study, or the effects may emerge under pathological contexts. These hypotheses should be tested in future studies.

Comparative overview of the effects of aerobic and resistance exercise on anxiety-like behavior, cognitive flexibility, and hippocampal synaptic plasticity parameters in healthy rats

Clinical studies show that physical exercise has anxiolytic and pro-cognitive properties for both healthy individuals and psychiatric patients. Most of these data refer to the effects of aerobic exercise. However, other modalities such as resistance exercise deserve more attention because they may also modulate brain function. This study aimed to compare the effects of an aerobic exercise protocol on a treadmill and a resistance exercise protocol on a ladder apparatus on anxiety-like behavior, cognitive flexibility, and neuroplasticity parameters in healthy animals. Adult male Wistar rats were divided into three groups: sedentary control, aerobic training, and resistance training. Subsequently, they were evaluated in the elevated plus-maze (EPM), light-dark box, and modified hole board (mHB) tests. The expressions of synaptophysin and postsynaptic plasticity protein 95 in the dorsal and ventral hippocampus were analyzed by immunofluorescence. The results demonstrated an anxiolytic effect promoted by exercise in the EPM, particularly in the animals submitted to aerobic training, and a mild pro-learning effect of both exercise modalities was observed in the mHB test. All groups showed similar outcomes in the other evaluations. Therefore, the exercise modalities investigated in the present study did not provide considerable modifications to such aspects of the emotional/cognitive functions and neuroplasticity under physiological contexts. Perhaps the two types of exercise acted in neurobiological pathways not analyzed in this study, or the effects may emerge under pathological contexts. These hypotheses should be tested in future studies.

Portuguese recommendations for the prevention, diagnosis and management of primary osteoporosis - 2018 update.

BACKGROUND: Advances in osteoporosis (OP)case definition, treatment options, optimal therapy duration and pharmacoeconomic evidence in the national context motivated the Portuguese Society of Rheumatology (SPR) to update the Portuguese recommendations for the diagnosis and management of osteoporosis published in 2007. METHODS: SPR bone diseases' working group organized meetings involving 55 participants (rheumatologists, rheumatology fellows and one OP specialist nurse) to debate and develop the document. First, the working group selected 11 pertinent clinical questions for the diagnosis and management of osteoporosis in standard clinical practice. Then, each question was investigated through literature review and draft recommendations were built through consensus. When insufficient evidence was available, recommendations were based on experts' opinion and on good clinical practice. At two national meetings, the recommendations were discussed and updated. A draft of the recommendations full text was submitted to critical review among the working group and suggestions were incorporated. A final version was circulated among all Portuguese rheumatologists before publication and the level of agreement was anonymously assessed using an online survey. RESULTS: The 2018 SPR recommendations provide comprehensive guidance on osteoporosis prevention, diagnosis, fracture risk assessment, pharmacological treatment initiation, therapy options and duration of treatment, based on the best available evidence. They attained desirable agreement among Portuguese rheumatologists. As more evidence becomes available, periodic revisions will be performed. Target audience and patient population: The target audience for these guidelines includes all clinicians. The target patient population includes adult Portuguese people. Intended use: These recommendations provide general guidance for typical cases. They may not be appropriate in all situations - clinicians are encouraged to consider this information together with updated evidence and their best clinical judgment in individual cases.

The occurrence of the filarial nematode Dirofilaria repens in canine hosts from Maio Island, Cape Verde.

The prevalence of canine Dirofilaria infection in Maio Island (Cape Verde) was analysed by serology, morphological and molecular identification of the parasite species. Blood and sera were collected from 150 dogs and 80 cats aged over 6 months from various localities of the island. DNA was extracted from blood and samples were screened by polymerase chain reaction (PCR) using microfilaria-specific primers. No Dirofilaria immitis was found in dogs while D. repens microfilariae were found in 5.3% of dogs and 6% were positive by PCR. The species identity was confirmed by sequencing of PCR products, which showed almost 100% homology with D. repens European sequences published in GenBank. No difference in Dirofilaria infection was observed between males and females or in dogs with different weights. However, older dogs and those from the western part of Maio Island were more frequently infected. No Dirofilaria was found in cats. This study represents the first evidence of D. repens in Cape Verde (West Africa) and highlights the need for implementing control measures and for a better surveillance of dirofilariosis in Africa.

Molecular and epidemiologic study of Clostridium difficile reveals unusual heterogeneity in clinical strains circulating in different regions in Portugal.

Clostridium difficile infection (CDI) represents a great healthcare burden in developed countries. The emergence of the epidemic PCR ribotype (RT) 027 and its acquired fluoroquinolones resistance have accentuated the need for an active surveillance of CDI. Here we report the first countrywide study of CDI in Portugal with the characterization of 498 C. difficile clinical isolates from 20 hospitals in four regions in Portugal regarding RT, virulence factors and antimicrobial susceptibility. We identified 96 RTs with marked variations between and within regions, as only six RTs appeared in all four regions. RT027 was the most frequent RT overall (18.5%) and among healthcare facility-associated isolates (19.6%), while RT014 was the most common among community-associated isolates (12%). The north showed a high RT diversity among isolates and a low moxifloxacin (MXF) resistance rate (11.9%), being the only region in which RT027 was not predominant. In contrast, the isolates from the centre presented the highest RT027 frequency, and 53.4% were resistant to MXF. Overall, MXF resistance (33.2%) was associated (p <0.001) with the presence of binary toxin genes and mutations in tcdC regardless of the RT. Both traits appeared in almost 30% of the strains. RT027 showed a reduced susceptibility to metronidazole (p <0.01), and RT126 had higher minimum inhibitory concentrations to vancomycin (p = 0.03) compared to other RTs. The present study highlights an unusual heterogeneity of RTs in Portugal, with a high frequency of hypervirulent RTs and the emergence of virulence factors in non-027 RTs, emphasizing the need for a surveillance system for CDI in Portugal.

The lysis cassette of DLP12 defective prophage is regulated by RpoE.

Expression of the lysis cassette (essD, ybcT, rzpD/rzoD) from the defective lambdoid prophage at the 12th minute of Escherichia coli's genome (DLP12) is required in some strains for proper curli expression and biofilm formation. Regulating production of the lytic enzymes encoded by these genes is critical for maintaining cell wall integrity. In lambdoid phages, late-gene regulation is mediated by the vegetative sigma factor RpoD and the lambda antiterminator Qλ. We previously demonstrated that DLP12 contains a Q-like protein (QDLP12) that positively regulates transcription of the lysis cassette, but the sigma factor responsible for this transcription initiation remained to be elucidated. In silico analysis of essDp revealed the presence of a putative - 35 and - 10 sigma site recognized by the extracytoplasmic stress response sigma factor, RpoE. In this work, we report that RpoE overexpression promoted transcription from essDp in vivo, and in vitro using purified RNAP. We demonstrate that the - 35 region is important for RpoE binding in vitro and that this region is also important for QDLP12-mediated transcription of essDp in vivo. A bacterial two-hybrid assay indicated that QDLP12 and RpoE physically interact in vivo, consistent with what is seen for Qλ and RpoD. We propose that RpoE regulates transcription of the DLP12 lysis genes through interaction with QDLP12 and that proper expression is dependent on an intact - 35 sigma region in essDp. This work provides evidence that the unique Q-dependent regulatory mechanism of lambdoid phages has been co-opted by E. coli harbouring defective DLP12 and has been integrated into the tightly controlled RpoE regulon.

Clinicopathological significance of immunoexpression of claudin-1 and claudin-7 in feline mammary carcinomas.

Claudins (CLDNs) are tight junction proteins that have a role in regulating cell adhesion and polarity, paracellular permeability, proliferation and differentiation. Several immunohistochemical studies have shown reduced expression of CLDN-1 and CLDN-7 in human and canine mammary carcinomas, suggesting that these proteins may participate in mammary carcinogenesis, invasion and metastasis. The present study characterizes expression of CLDN-1 and CLDN-7 in feline mammary carcinomas (n = 52) and their metastases (n = 29). There was an inverse association between CLDN-7 expression and histological grade of tumour. Reduced expression of CLDN-7 was significantly associated with decreased tubule formation, high proliferative activity and metastasis. No significant associations were found between CLDN-1 expression and any of these features. Evaluation of expression of CLDN-7, but not CLDN-1, may therefore provide prognostic information, assisting in the diagnosis of a subgroup of aggressive feline mammary carcinomas that share some features with the recently described 'claudin-low' subgroup of human breast cancer.

Reduced expression of claudin-2 is associated with high histological grade and metastasis of feline mammary carcinomas.

Claudins (CLDNs) are a family of tight junction (TJ) proteins that play an important role in maintaining cell polarity, in controlling paracellular ion flux and in regulating cell proliferation and differentiation. There is a growing body of evidence that associates changes in CLDN expression with the development of human breast cancer. In the present study CLDN-2 expression was examined immunohistochemically in samples of normal feline mammary tissue (n = 5) and mammary carcinomas (n = 52), including metastatic lesions (n = 29). Seventy-seven percent of carcinomas showed reduced CLDN-2 expression compared with that observed in normal mammary gland. Reduced expression of CLDN-2 was significantly associated with a high histological grade of carcinoma (P = 0.011), with 88.6% of grade II/III carcinomas showing decreased expression. Furthermore, CLDN-2 down-regulation was significantly associated with metastatic disease (P = 0.0027), with 93.1% of cases with signs of metastasis showing decreased expression of this protein. CLDN-2 may constitute a molecular marker for identification of a subgroup of feline mammary carcinomas characterized by high histological grade and the development of metastasis.

A Q-like transcription factor regulates biofilm development in Escherichia coli by controlling expression of the DLP12 lysis cassette.

The DLP12 lysis cassette (essD, ybcT, rzpD/rzoD) is required in certain Escherichia coli strains for normal curli expression and biofilm development. Tightly controlled regulation of the lysis cassette is of particular importance, since its overexpression causes host cell lysis. In silico analysis revealed a putative intrinsic transcriptional terminator 100 bp upstream of essD and within 2000 bp of ybcQ (Q(DLP12)), a putative lambda (λ) Q-like antiterminator. We hypothesized that Q(DLP12) may be required for effective expression of the lysis cassette. In this work we report on the role of Q(DLP12) as a positive regulator of DLP12 lysis cassette expression. Mutants lacking Q(DLP12) exhibited a biofilm-defective phenotype analogous to that of the lysis cassette knockouts. This defect occurred through the downregulation of curli transcription, which is also consistent with that seen in the lysis cassette mutants and was restored by complementation by ectopic expression of Q(DLP12). In addition, Q(DLP12) overexpression caused cell lysis, as demonstrated by leakage of ß-galactosidase activity from cells. This was accompanied by upregulation of the DLP12 lysis cassette as demonstrated by increased essD transcription, which was documented with gfp-reporter assays, RT-PCR and chromatin immunoprecipitation (ChIP). We provide evidence that this Q-mediated effect resulted from direct interaction of Q(DLP12) with the lysis cassette promoter (essDp), as demonstrated by electrophoretic gel mobility shift assay (EMSA). We propose that Q(DLP12) encodes a functional transcriptional regulator, which promotes expression of the DLP12 lysis cassette. This work provides evidence of a regulator from a defective prophage affecting host cell physiology.

The role of ionic interactions in the adherence of the Staphylococcus epidermidis adhesin SdrF to prosthetic material.

Staphylococcus epidermidis infections are common complications of prosthetic device implantation. SdrF, a surface protein, appears to play a critical role in the initial colonization step by adhering to type I collagen and Dacron™. The role of ionic interactions in S. epidermidis adherence to prosthetic material was examined. SdrF was cloned and expressed in Lactococcus lactis. The effect of pH, cation concentration, and detergents on adherence to different types of plastic surfaces was assessed by crystal violet staining and bacterial cell counting. SdrF, in contrast with controls and other S. epidermidis surface proteins, bound to hydrophobic materials such as polystyrene. Binding was an ionic interaction and was affected by surface charge of the plastic, pH, and cation concentration. Adherence of the SdrF construct was increased to positively charged plastics and was reduced by increasing concentrations of Ca(2+) and Na(+). Binding was optimal at pH 7.4. Kinetic studies demonstrated that the SdrF B domain as well as one of the B subdomains was sufficient to mediate binding. The SdrF construct also bound more avidly to Goretex™ than the lacotococcal control. SdrF is a multifunctional protein that contributes to prosthetic devices infections by ionic, as well as specific receptor-ligand interactions.

Manchester triage in acute pulmonary embolism: can it unmask the grand impersonator?

BACKGROUND: Acute pulmonary embolism (PE) is associated with high mortality risk. Early diagnosis is difficult because of non-specific clinical presentation and delay in imaging confirmation. Manchester Triage (MT) prioritises patients on the basis of illness severity and potentially recognises those with higher mortality risk. No studies of the role and impact of MT on rapid PE diagnosis and in-hospital mortality (IHM) have been carried out. OBJECTIVE: To assess the appropriateness of MT in this set of patients presenting acutely to the emergency department (ED), and to determine whether it assists in a rapid diagnosis, acts as a protective triage tool and affects short-term mortality. METHODS: Single-centre retrospective study of 176 consecutive patients with PE, assessed by MT in the ED between January 2006 and October 2010 (mean age 70.5±15.7 years, 38.6% men). The primary outcome measure was all-cause IHM. RESULTS: IHM was seen in 30 (17%) patients. More than half of the patients with PE (54%) were classified as target time for first medical observation (MOb) ≤10 min. 73.3% of IHM occurred in this group (p=0.020) with several increased markers of illness severity. MOb ≤10 min was not associated with faster PE imaging confirmation. The average door-to-diagnosis time (PEDx) was 26.8±36.8 h and PEDx >17.0 h was associated with higher IHM (p=0.017). On multivariate analysis, thrombolysis and MOb ≤10 min were included in an IHM predictor model. CONCLUSION: MT has high sensitivity in identifying patients with PE at risk. Those patients assigned as MOb ≤10 min have increased markers of illness severity and higher IHM. MT acts as a protective system in this challenging set and should be used as a patient's first assessment, aiding the emergency medical team to recognise those in need of urgent assessment and treatment.

Draft genome sequences of two Pseudomonas aeruginosa clinical isolates with different antibiotic susceptibilities.

Pseudomonas aeruginosa is a primary cause of opportunistic infections. We have sequenced and annotated the genomes of two P. aeruginosa clinical isolates evidencing different antibiotic susceptibilities. Registered differences in the composition of their accessory genomes may provide clues on P. aeruginosa strategies to thrive in different environments like infection loci.

Reuma.pt - the rheumatic diseases portuguese register.

INTRODUCTION: Since June 2008, Portuguese rheumatologists have been collecting on a routine basis, data into the nationwide Reuma.pt, the Rheumatic Diseases Portuguese Register from the Portuguese Society of Rheumatology (SPR), which includes rheumatic patients (rheumatoid arthritis - RA, ankylosing spondylitis - AS, psoriatic arthritis - PsA and juvenile idiopathic arthritis - JIA) receiving biological therapies or patients receiving synthetic disease modifying anti-rheumatic drugs (DMARDs). The aim of this publication is to describe the structure of Reuma.pt and the population registered since June 2008. METHODS: Demographic and anthropometric data, life style habits, work status, co-morbidities, disease activity and functional assessment scores, previous and current therapies, adverse events codified by the Medical Dictionary for Regulatory Activities (MedDRA), reasons for discontinuation and laboratory measurements are registered at each visit. The platform is based on a structured electronic medical record linked to a SQL Server database. All Rheumatology Departments assigned to the Portuguese National Health Service (n=21), 2 Military Hospitals (Lisboa and Porto), 1 public-private Institution and 6 private centers adhered to the Register. Until now, 18 centers have entered data into Reuma.pt. RESULTS: By January 2011, 3438 patients and 16130 visits had been registered. 2162 (63%) were RA patients, 700 of them treated with biological agents and 1462 with synthetic DMARDs. From the 515 (15%) AS patients, 297 were medicated with biological and 218 with non-biological therapies. 293 (8%) were PsA patients, 151 treated with biological drugs and 142 with other treatment strategies. 368 (11%) had the diagnosis of JIA, 68 were under biological treatment and 300 were managed with other treatment options. The register also includes 100 (3%) patients with other rheumatic diseases, submitted to treatments that required hospital day care infusions including 18 exposed to biological therapies. CONCLUSIONS: Registers are crucial to ensure correct clinical use, adequate assessment of post-marketing biological therapies' efficacy and safety, thus contributing for a better cost-benefit ratio. Reuma.pt, is a powerful and accurate tool to answer to these unmet needs. It presents a national coverage of the rheumatology centers and constitutes an invaluable resource for scientific research and to improve rheumatic patients care.

Role of DLP12 lysis genes in Escherichia coli biofilm formation.

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz(1)). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of (14)C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz(1) gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

Role of biofilm in Staphylococcus aureus and Staphylococcus epidermidis ventricular assist device driveline infections.

OBJECTIVE: Infections, especially those involving drivelines, are among the most serious complications that follow ventricular assist device implantation. Staphylococci are the most common causes of these infections. Once driveline infections are established, they can remain localized or progress as an ascending infection to cause metastatic seeding of other tissue sites. Although elaboration of biofilm appears to be critical in prosthetic device infections, its role as a facilitator of staphylococcal infection and migration along the driveline and other prosthetic devices is unclear. METHODS: A murine model of driveline infection was used to investigate staphylococcal migration along the driveline. A biofilm-producing strain of Staphylococcus epidermidis and a Staphylococcus aureus strain and its intercellular adhesion gene cluster (ica)-negative (biofilm-deficient) isogenic mutant were used in these studies. Bacterial density on the driveline and the underlying tissue was measured over time. Scanning electron microscopy was used to examine the morphology of S epidermidis biofilm formation as the infection progressed. RESULTS: The biofilm-deficient S aureus mutant was less effective at infecting and migrating along the driveline than the wild-type strain over time. However, the ica mutation had no effect on the ability of the strain to infect underlying tissue. S aureus exhibited more rapid migration than S epidermidis. Scanning electron microscopy revealed the deposition of host matrix on the Dacron material after implantation. This was followed by elaboration of a bacterial biofilm that correlated with more rapid migration along the driveline. CONCLUSIONS: Biofilm formation is a critical virulence determinant that facilitates the progression of drivelines infections.