Objectives: To study the occurrence of antimicrobial resistance among common bacterial pathogens from dogs and relate resistance patterns to data on consumption of antimicrobials. Methods:The antimicrobial susceptibility patterns of 201 Staphylococcus intermedius, 37 Streptococcus canis, 39 Pseudomonas aeruginosa, 25 Pasteurella multocida, 29 Proteus spp. and 449 Escherichia coli isolates from clinical submissions from dogs were determined by a broth-dilution method for determination of minimal inhibitory concentration. Data for consumption of antimicrobials were retrieved from VetStat, a national database for reporting antimicrobial prescriptions.Results: The majority of the antimicrobials prescribed for dogs were broad-spectrum compounds, and extended-spectrum penicillins, cephalosporins and sulphonamides 1 trimethoprim together accounted for 81% of the total amount used for companion animals. Resistance to cephalosporins and amoxicillin with clavulanic acid was very low for all bacterial species examined, except for P. aeruginosa, and resistance to sulphonamides and trimethoprim was low for most species. Among the S. intermedius isolates, 60.2% were resistant to penicillin, 30.2% to fusidic acid and 27.9% to macrolides. Among E. coli isolates, the highest level of resistance was recorded for ampicillin, sulphonamides, trimethoprim, tetracyclines and streptomycin. Certain differences in resistance patterns between isolates from different sites or organs were noticed for E. coli, S. intermedius and Proteus isolates.Conclusions: This investigation provided data on occurrence of antimicrobial resistance in important pathogenic bacteria from dogs, which may be useful for the small animal practitioner. Resistance was low to the compounds that were most often used, but unfortunately, these compounds were broadspectrum. Data on resistance and usage may form a background for the establishment of a set of recommendations for prudent use of antimicrobials for companion animals.

Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.

SummaryBackgroundIntensive antiplatelet therapy with three agents might be more effective than guideline treatment for preventing recurrent events in patients with acute cerebral ischaemia. We aimed to compare the safety and efficacy of intensive antiplatelet therapy (combined aspirin, clopidogrel, and dipyridamole) with that of guideline-based antiplatelet therapy.MethodsWe did an international, prospective, randomised, open-label, blinded-endpoint trial in adult participants with ischaemic stroke or transient ischaemic attack (TIA) within 48 h of onset. Participants were assigned in a 1:1 ratio using computer randomisation to receive loading doses and then 30 days of intensive antiplatelet therapy (combined aspirin 75 mg, clopidogrel 75 mg, and dipyridamole 200 mg twice daily) or guideline-based therapy (comprising either clopidogrel alone or combined aspirin and dipyridamole). Randomisation was stratified by country and index event, and minimised with prognostic baseline factors, medication use, time to randomisation, stroke-related factors, and thrombolysis. The ordinal primary outcome was the combined incidence and severity of any recurrent stroke (ischaemic or haemorrhagic; assessed using the modified Rankin Scale) or TIA within 90 days, as assessed by central telephone follow-up with masking to treatment assignment, and analysed by intention to treat. This trial is registered with the ISRCTN registry, number ISRCTN47823388.Findings3096 participants (1556 in the intensive antiplatelet therapy group, 1540 in the guideline antiplatelet therapy group) were recruited from 106 hospitals in four countries between April 7, 2009, and March 18, 2016. The trial was stopped early on the recommendation of the data monitoring committee. The incidence and severity of recurrent stroke or TIA did not differ between intensive and guideline therapy (93 [6%] participants vs 105 [7%]; adjusted common odds ratio [cOR] 0·90, 95% CI 0·67–1·20, p=0·47). By contrast, intensive antiplatelet therapy was associated with more, and more severe, bleeding (adjusted cOR 2·54, 95% CI 2·05–3·16, p<0·0001).InterpretationAmong patients with recent cerebral ischaemia, intensive antiplatelet therapy did not reduce the incidence and severity of recurrent stroke or TIA, but did significantly increase the risk of major bleeding. Triple antiplatelet therapy should not be used in routine clinical practice.FundingNational Institutes of Health Research Health Technology Assessment Programme, British Heart Foundation.

Increasing evidence suggests that the water/glycerol channel aquaporin-3 (AQP3) plays a pivotal role in cancer metastasis. AQP3 knockout mice were resistant to skin tumor formation and overexpression correlated with metastasis and poor prognosis in patients with breast or gastric cancer. In cultured cancer cells, increased AQP3 expression stimulated several intracellular signaling pathways and resulted in increased cell proliferation, migration, and invasion as well as aggravation of epithelial-to-mesenchymal transition. Besides AQP facilitated water transport at the leading edge of migrating cells, AQP3 signaling mechanisms are beginning to be unraveled. Here, we give a thorough review of current knowledge regarding AQP3 expression in cancer and how AQP3 contributes to cancer progression via signaling that modulates cellular mechanisms. This review article will expand our understanding of the known pathophysiological findings regarding AQP3 in cancer.

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To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O 2 , with parallel controls cultured in atmospheric air. Both were enriched with 5% CO 2 . The results showed a dramatic increase in the growth of C. pneumoniae but not of C. trachomatis.The chlamydial developmental cycle is biphasic, alternating between an infectious metabolically inactive elementary body (EB) specialized for extracellular survival and a noninfectious proliferating intracellular reticulate body (RB). During the course of an infection, the EB is endocytosed by a susceptible host cell into a host-derived vacuole, the chlamydial inclusion. After internalization, the EB develops into an RB, which proliferates by binary fission. Following several rounds of proliferation lasting 48 to 72 h, RBs transform into EBs and are released by the disruption of the host cell (for a review, see reference 9).Chlamydia trachomatis is an obligate human pathogen causing ocular and genital infections. Chlamydia pneumoniae causes respiratory tract infections, often asymptomatic, but may cause bronchitis and pneumonia (5). Traditionally, both C. trachomatis and C. pneumoniae have been studied in vitro by infecting cell culture monolayers and incubating the infected cells in incubators in a humid atmosphere containing atmospheric air enriched with 5% CO 2 , resulting in an oxygen concentration of approximately 20%. However, the in vivo oxygen tension is much lower, generally in the range of 3 to 6% (Table 1), and as different tissues have different oxygen requirements, the in vivo oxygen tension may vary considerably from tissue to tissue. The oxygen requirements of Chlamydia have never been evaluated before, but it is known that the oxygen tension of host tissue is important for viral replication and the viral life cycle (3) and that many infecting microorganisms are microaerophilic. As Chlamydia proliferates in vivo where the oxygen tension varies between different tissues, it is plausible that Chlamydia is also affected by the oxygen tension and would experience enhanced growth in tissues with optimum oxygen tension.C. trachomatis and C. pneumoniae produced enlarged inclusions in 4% oxygen. To determine the visible effect of low oxygen tension on chlamydial inclusions, infected HeLa cells were cultured on coverslips at 4% and 20% O 2 . HeLa 229 cells (ATCC, Rockville, MD) cultured in 24-well trays (TPP, Trasadingen, Switzerland) were infected with either C. trachomatis serovar D/UW-3/CX (ATCC) or C. pneumoniae CWL029 (ATCC) as previously described (12, 15) and cultured in the presence of cycloheximide in either 4% or 20% oxygen atmospheres. Low-oxygen atmospheres were achieved by placing trays with infected HeLa cells in an airtight custom-made box (40 by 30 by 16 cm [width by diameter by height]). An OX-500 Clark-type oxygen sensor (UniSense, Aarhus, Denmark) was placed inside the box to measure the oxygen concentration. The box was flushed w...

Plasticity of epithelial cell‐cell adhesion is vital in epithelial homeostasis and is regulated in multiple processes associated with cell migration, such as embryogenesis and wound healing. In cancer, cell‐cell adhesion is compromised and is associated with increased cell migration and metastasis. Aquaporin (AQP) water channels facilitate water transport across cell membranes and are essential in the regulation of body water homeostasis. Increased expression of several AQPs, especially AQP5, is associated with increased cancer cell migration, metastasis, and poor prognosis. We found that AQP5 overexpression in normal epithelial cells induced cell detachment and dissemination from migrating cell sheets. AQP5 reduced both cell‐cell coordination during collective migration and overall distance covered by the migrating cell sheets. AQP5 and the isoforms AQP1 and AQP4 decreased, whereas AQP3 increased, levels of plasma membrane‐associated lateral junctional proteins. This regulation was mediated by the cytoplasmic domains of the AQPs. This shows that the AQPs have dual functions in epithelial physiology: as channel proteins and as differential regulators of cell‐cell adhesiveness. This regulation may contribute to dynamic regulation of cell junctions in processes such as embryogenesis and wound healing and also explain the pivotal roles of AQPs in carcinogenesis and metastasis.—Login, F. H., Jensen, H. H., Pedersen, G. A., Koffman, J. S., Kwon, T.‐H., Parsons, M., Nejsum, L. N. Aquaporins differentially regulate cell‐cell adhesion in MDCK cells. FASEB J. 33, 6980–6994 (2019). http://www.fasebj.org

Prolactin (PRL) and its receptor (PRLR) are implicated in breast cancer invasiveness, although their exact roles remain controversial. The Na(+)/H(+) exchanger (NHE1) plays essential roles in cancer cell motility and invasiveness, but the PRLR and NHE1 have not previously been linked. Here we show that in T47D human breast cancer cells, which express high levels of PRLR and NHE1, exposure to PRL led to the activation of Janus kinase-2 (JAK2)/signal transducer and activator of transcription-5 (STAT5), Akt, and ERK1/2 signaling and the rapid formation of peripheral membrane ruffles, known to be associated with cell motility. NHE1 was present in small ruffles prior to PRL treatment and was further recruited to the larger, more dynamic ruffles induced by PRL exposure. In PRL-induced ruffles, NHE1 colocalized with activated Akt, ERK1/2, and the ERK effector p90Ribosomal S kinase (p90RSK), known regulators of NHE1 activity. Stimulation of T47D cells with PRL augmented p90RSK activation, Ser703-phosphorylation of NHE1, NHE1-dependent intracellular pH recovery, pericellular acidification, and NHE1-dependent invasiveness. NHE1 activity and localization to ruffles were attenuated by the inhibition of Akt and/or ERK1/2. In contrast, noncancerous MCF10A breast epithelial cells expressed NHE1 and PRLR at lower levels than T47D cells, and their stimulation with PRL induced neither NHE1 activation nor NHE1-dependent invasiveness. In conclusion, we show for the first time that PRLR activation stimulates breast cancer cell invasiveness via the activation of NHE1. We propose that PRL-induced NHE1 activation and the resulting NHE1-dependent invasiveness may contribute to the metastatic behavior of human breast cancer cells.