Pyridinyl 4-(2-oxoalkylimidazolidin-1-yl)benzenesulfonates and their hydrochloride salts as novel water soluble antimitotic prodrugs bioactivated by cytochrome P450 1A1 in breast cancer cells.

We developed first-in-class antimitotic prodrugs phenyl 4-(2-oxo-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) bioactivated by cytochrome P450 (CYP) 1A1 that are highly selective toward several breast cancer cells. However, they show sparingly water solubility. Therefore, we replaced their phenyl ring B with a substituted pyridinyl group preparing novel pyridinyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PYRAIB-SOs) and their hydrochloride salts. Our results evidence that PYRAIB-SO hydrochloride salts show higher water solubility compared to their neutral and PAIB-SO counterparts by up to 625-fold. PYRAIB-SOs with a nitrogen atom at position 3 of the pyridinyl ring exhibited strong antiproliferative activity (IC50: 0.03-3.3 µM) and high selectivity (8->1250) toward sensitive CYP1A1-positive breast cancer cells and cells stably transfected with CYP1A1. They induce cell cycle arrest in the G2/M phase and disrupt microtubule dynamic assembly. Enzymatic assays confirmed that CYP1A1 metabolizes PYRAIB-SOs into their active form with in vitro hepatic half-lives (55-120 min) in rodent and human liver microsomes. Overall, this will allow to increase drug concentration for in vivo studies.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Prevalence and Antimicrobial Resistance of Salmonella Dublin and Thermotolerant Campylobacter in Liver from Veal Calves in Québec, Canada.

Salmonella Dublin and Campylobacter spp. are two foodborne pathogens of importance. A small number of studies reported that consumption of veal liver was associated with an increased risk of human illness from these two pathogens. To better characterize the risk of exposure from liver, a cross-sectional study was conducted to estimate the prevalence of white veal calf liver contamination with these two pathogens and to characterize the antimicrobial non-susceptibility patterns of isolates. Veal liver samples were collected at two slaughterhouses in Quebec, Canada, in 2016 and 2017. Samples were submitted for polymerase chain reaction (PCR) screening followed by culture of Salmonella and thermotolerant Campylobacter. Isolates were tested for antimicrobial susceptibility using broth microdilution. Salmonella Dublin was the only serotype cultured from 3.6% (95% confidence interval [CI]: 0.0-7.9) of 560 liver samples. Among them and for technical reasons, 498 were tested by PCR for Campylobacter. The prevalence of PCR-positive livers was estimated to be 65.8% (95% CI: 58.7-72.9) for Campylobacter jejuni and 7.0% (95% CI: 3.9-10.1%) for Campylobacter coli. Fourteen Salmonella Dublin isolates were submitted for antimicrobial resistance (AMR) testing; all were non-susceptible to at least eight antimicrobials from six different classes. Most (81.4%) of the 188 C. jejuni isolates submitted for AMR testing were non-susceptible to tetracycline, and 23.0% of isolates were non-susceptible to nalidixic acid and ciprofloxacin. Of the seven C. coli isolates, four were multidrug resistant. This study highlights the importance of veal liver as a potential source of exposure to multidrug-resistant Salmonella Dublin and thermotolerant Campylobacter spp.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide salts: Novel hydrosoluble prodrugs of antimitotics selectively bioactivated by the cytochrome P450 1A1 in breast cancer cells.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs) are members of a new family of prodrugs bioactivated by cytochrome P450 1A1 (CYP1A1) in breast cancer cells into their potent 4-(2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide metabolites (PIB-SAs). One of the predominant problems for the galenic formulation and administration of PAIB-SAs in animal studies is their poor hydrosolubility. To circumvent that difficulty, we report the design, the synthesis, the chemical characterization, the evaluation of the aqueous solubility, the antiproliferative activity and the mechanism of action of 18 new Na+, K+ and Li+ salts of PAIB-SAs. Our results evidenced that the latter exhibited highly selective antiproliferative activity toward MCF7 and MDA-MB-468 breast cancer cells expressing endogenously CYP1A1 compared to insensitive MDA-MB-231 and HaCaT cells. Moreover, PAIB-SA salts 1-18 are significantly more hydrosoluble (3.9-9.4 mg/mL) than their neutral counterparts (< 0.0001 mg/mL). In addition, the most potent PAIB-SA salts 1-3 and 10-12 arrested the cell cycle progression in the G2/M phase and disrupted the cytoskeleton's dynamic assembly. Finally, PAIB-SA salts are N-dealkylated by CYP1A1 into their corresponding PIB-SA metabolites, which are potent antimitotics. In summary, our results show that our water-soluble PAIB-SA salts, notably the sodium salts, still exhibit potent antiproliferative efficacy and remain prone to CYP1A1 bioactivation. In addition, these PAIB-SA salts will allow the development of galenic formulations suitable for further biopharmaceutical and pharmacodynamic studies.

Estimation of the accuracy of an ELISA test applied to bulk tank milk for predicting herd-level status for Salmonella Dublin in dairy herds using Bayesian Latent Class Models.

Enzyme-Linked Immunosorbent Assay (ELISA) test is commonly used for detection of antibodies to Salmonella Dublin in individual bovine milk samples. However, little is known about its accuracy when used on bulk tank milk for determining herd-level S. Dublin status and when evaluated without assuming a perfect reference test. The objectives of this study were: i) to estimate the herd prevalence of S. Dublin among dairy cattle herds in Québec, Canada; ii) to estimate the herd sensitivity and specificity of a commercially available ELISA test when used on bulk milk; iii) to examine how the diagnostic test accuracy varies with different bulk milk ELISA cut-offs; and (iv) to assess the added value of combining ELISA screening of bulk milk and individual serum of 10 animals for determining S. Dublin herd status. A cohort of 302 dairy herds selected in three regions (population 1) and 58 herds that have already tested positive to S. Dublin (population 2) were recruited. A total of 715 bulk milk samples and 7150 individual blood samples from cattle over 3 months old (10 animals per herd) sampled on two occasions were collected. Testing was conducted using PrioCHECK™ Salmonella Ab bovine Dublin ELISA test for milk (Bmilk ELISA: test under investigation) and for serum of 10 individual animals (Serum10 ELISA: imperfect reference test) to determine the herd-level S. Dublin status. A latent class model for two populations, two tests, allowing for conditional dependence between tests was fit within a Bayesian framework. At cut-off PP % ≥ 15 for a Bmilk ELISA, which is used by provincial authorities, the herd prevalence of S. Dublin estimated using informative prior was 6.8 % (4.3-9.9) in population 1. The herd sensitivity and specificity estimates (95 % Bayesian Credibility Intervals) for Bmilk ELISA were 40.6 % (15.6-88.8) and 91.9 % (88.3-95.8), respectively. Positive and negative predictive values of Bmilk ELISA applied in population 1 were 26.4 % (8.5-60.2) and 95.8 % (92.1-99.2), respectively. Increasing Bmilk ELISA cut-offs had little influence on predictive values. The combination of both ELISA tests did not improve the diagnostic accuracy of S. Dublin. Our study shows that a test-positive herd based on a single bulk milk sample would require complementary tests for status confirmation. However, a test-negative herd could be classified as true negative with a high certainty.

Mitochondrial matrix-localized Src kinase regulates mitochondrial morphology.

The architecture of mitochondria adapts to physiological contexts: while mitochondrial fragmentation is usually associated to quality control and cell death, mitochondrial elongation often enhances cell survival during stress. Understanding how these events are regulated is important to elucidate how mitochondrial dynamics control cell fate. Here, we show that the tyrosine kinase Src regulates mitochondrial morphology. Deletion of Src increased mitochondrial size and reduced cellular respiration independently of mitochondrial mass, mitochondrial membrane potential or ATP levels. Re-expression of Src targeted to the mitochondrial matrix, but not of Src targeted to the plasma membrane, rescued mitochondrial morphology in a kinase activity-dependent manner. These findings highlight a novel function for Src in the control of mitochondrial dynamics.

Bayesian accuracy estimates of environmental sampling for determining herd paratuberculosis infection status and its association with the within-herd individual fecal culture prevalence in Québec dairies.

The objectives of this retrospective analysis were to: 1) estimate the diagnostic sensitivity (Se) and specificity (Sp) of bacterial culture of environmental samples for determining Mycobacterium avium subsp. paratuberculosis (MAP) infection status in Québec dairies, using a Bayesian Latent Class Model (BLCM); and 2) determine the association between the number of positive environmental samples and the individual fecal culture (IFC) apparent and true MAP within-herd prevalence. Environmental and individual fecal samples were collected from 87 commercial dairy herds participating in previous research projects. Environmental samples included two composite samples of 20 g collected from different locations within each of the following sites: an area where manure from the majority of adult cattle accumulates, a manure storage area and another site of manure accumulation chosen by the veterinarian. Samples were cultured using the MGIT Para TB culture liquid media and the BACTEC MGIT 960 system. The Se and Sp of environmental sampling were estimated using a one-test-one-population BLCM. Herds were considered positive for environmental sampling if at least one out of the six samples collected was positive. The apparent and true IFC within-herd MAP prevalence estimates for each herd were obtained using a two-stage cluster BLCM, then merged in a single dataset with the environmental sample results. The association between the within-herd MAP prevalence results (apparent and true), and the number of positive environmental samples was assessed using a zero-inflated negative binomial (ZINB) model. In all BLCMs, median posterior estimates and 95 % Bayesian credible intervals (BCI) were obtained with OpenBUGS statistical freeware. Se and Sp of environmental sampling were 43.7 % (95 % BCI: 32.5-55.5) and 96.2 % (95 % BCI: 84.2-99.8), respectively. Overall, the number of positive environmental samples increased with the apparent and true MAP within-herd prevalence. The true prevalence was higher than the apparent prevalence for a given number of positive environmental samples. The probability of not observing a positive environmental sample decreased with the prevalence. Despite its imperfect accuracy, environmental sampling is an inexpensive and non-invasive sampling method to determine MAP infection status in tie-stall herds that can be used as a proxy to estimate the true within-herd prevalence. The absence of positive environmental samples in a single sampling visit is likely an indicator of a very low within-herd prevalence rather than being MAP exempt.

Erratum to: Neonatal Microsurgical Repair of a Congenital Abdominal Aortic Aneurysm with a Cadaveric Graft.

[This corrects the article DOI: 10.1055/s-0041-1723019.].

Evaluation of the feasibility and impacts of in situ simulation in emergency medicine-a mixed-method study protocol.

INTRODUCTION: In situ simulation (ISS) consists of performing a simulation in the everyday working environment with the usual team members. The feasibility of ISS in emergency medicine is an important research question, because ISS offers the possibility for repetitive, regular simulation training consistent with specific local needs. However, ISS also raises the issue of safety, since it might negatively impact the care of other patients in the emergency department (ED). Our hypothesis is that ISS in an academic high-volume ED is feasible, safe and associated with benefits for both staff and patients. METHODS: A mixed-method, including a qualitative method for the assessment of feasibility and acceptability and a quantitative method for the assessment of patients' safety and participants' psychosocial risks, will be used in this study.Two distinct phases are planned in the ED of the CHU de Québec-Université Laval (Hôpital de l'Enfant-Jésus) between March 2021 and October 2021. Phase 1: an ISS programme will be implemented with selected ED professionals to assess its acceptability and safety and prove the validity of our educational concept. The number of cancelled sessions and the reasons for cancellation will be collected to establish feasibility criteria. Semistructured interviews will evaluate the acceptability of the intervention. We will compare unannounced and announced ISS. Phase 2: the impact of the ISS programme will be measured with validated questionnaires for the assessment of psychosocial risks, self-confidence and perceived stress among nonselected ED professionals, with comparison between those exposed to ISS and those that were not. ETHICS AND DISSEMINATION: The CHU de Québec-Université Laval Research ethics board has approved this protocol (#2020-5000). Results will be presented to key professionals from our institution to improve patient safety. We also aim to publish our results in peer-reviewed journals and will submit abstracts to international conferences to disseminate our findings.

Neonatal Microsurgical Repair of a Congenital Abdominal Aortic Aneurysm with a Cadaveric Graft.

Congenital abdominal aortic aneurysms (AAA) are an extremely rare entity. We present the case of a female fetus diagnosed with an AAA on routine prenatal ultrasound. A postnatal computed tomography angiogram revealed an infrarenal AAA with a narrow proximal neck. Surgery was performed on day of life 14 using a cadaveric femoral artery graft. The proximal anastomosis was performed under the microscope given the severity of the aortic stenosis and the proximity of the renal arteries. The patient's postoperative course was uneventful and she is developing normally 1 year after surgery. The graft remains permeable, albeit with evidence of proximal and distal stenosis and graft calcification on imaging.

Postoperative outcomes in surgical COVID-19 patients: a multicenter cohort study.

BACKGROUND: Data on postoperative outcomes of the COVID-19 patient population is limited. We described COVID-19 patients who underwent a surgery and the pandemic impact on surgical activities. METHODS: We conducted a multicenter cohort study between March 13 and June 192,020. We included all COVID-19 patients who underwent surgery in nine centres of the Province of Québec, the Canadian province most afflicted by the pandemic. We also included concomitant suspected COVID-19 (subsequently confirmed not to have COVID-19) patients and patients who had recovered from it. We collected data on baseline characteristics, postoperative complications and postoperative mortality. Our primary outcome was 30-day mortality. We also collected data on overall surgical activities during this first wave and during the same period in 2019. RESULTS: We included 44 COVID-19 patients, 18 suspected patients, and 18 patients who had recovered from COVID-19 at time of surgery. Among the 44 COVID-19 patients, 31 surgeries (71%) were urgent and 16 (36%) were major. In these patients, pulmonary complications were frequent (25%) and 30-day mortality was high (15.9%). This mortality was higher in patients with symptoms (23.1%) compared to those without symptoms (5.6%), although not statistically significant (p = 0.118). Of the total 22,616 cases performed among participating centres during the study period, only 0.19% had COVID-19 at the time of surgery. Fewer procedures were performed during the study period compared to the same period in 2019 (44,486 cases). CONCLUSION: In this Canadian cohort study, postoperative 30-day mortality in COVID-19 patients undergoing surgery was high (15.9%). Although few surgeries were performed on COVID-19 patients, the pandemic impact on surgical activity volume was important. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04458337 .

Chart Audit of Spiritual Care Documentation: Continuous Quality Improvement.

Eight spiritual care practitioners at an acute care teaching hospital undertook a systematic chart audit of their documentation practices in the patient electronic health record. The purpose was to evaluate their practices using the standards of their professional association and regulatory college. A preliminary "mock audit" was essential for the overall success of the audit. Plans for ongoing chart audits will lead to continuous quality improvement. A limitation was that their manager acted as both improvement coach and performance evaluator.

Intramitochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells.

High levels and activity of Src kinase are common among breast cancer subtypes, and several inhibitors of the kinase are currently tested in clinical trials. Alterations in mitochondrial activity is also observed among the different types of breast cancer. Src kinase is localized in several subcellular compartments, including mitochondria where it targets several proteins to modulate the activity of the organelle. Although the subcellular localization of other oncogenes modulates the potency of known treatments, nothing is known about the specific role of intra-mitochondrial Src (mtSrc) in breast cancer. The aim of this work was to determine whether mtSrc kinase has specific impact on breast cancer cells. We first observed that activity of mtSrc is higher in breast cancer cells of the triple negative subtype. Over-expression of Src specifically targeted to mitochondria reduced mtDNA levels, mitochondrial membrane potential and cellular respiration. These alterations of mitochondrial functions led to lower cellular viability, shorter cell cycle and increased invasive capacity. Proteomic analyses revealed that mtSrc targets the mitochondrial single-stranded DNA-binding protein, a regulator of mtDNA replication. Our findings suggest that mtSrc promotes aggressiveness of breast cancer cells via phosphorylation of mitochondrial single-stranded DNA-binding protein leading to reduced mtDNA levels and mitochondrial activity. This study highlights the importance of considering the subcellular localization of Src kinase in the development of potent therapy for breast cancer.

Oral Lorazepam is not Superior to Placebo for Lowering Stress in Children Before Digestive Endoscopy: A Double-Blind, Randomized, Controlled Trial.

BACKGROUND: Digestive endoscopies must be performed within a safe and comfortable environment. We have previously shown that the quality of intravenous sedation is influenced by preoperative stress. AIM: Our primary objective was to compare the effects of oral lorazepam and placebo on the salivary cortisol response of children undergoing a digestive endoscopy. Secondary objectives were the assessment of procedural pain and comfort as well as the occurrence of adverse events. METHODS: Participants were randomized and received either lorazepam, placebo, or no premedication. Saliva was collected upon arrival at the hospital and 1 h following randomization. The sedation protocol included midazolam and fentanyl ± ketamine. Procedural pain was evaluated with the Nurse Assessed Patient Comfort Score (NAPCOMS). Patients completed a postoperative questionnaire. The primary outcome was defined as the proportion of children having a cortisol decrease ≥ 15 nmol/L. RESULTS: 101 participants (54 females) were included. The rate of children having a cortisol decrease ≥ 15 nmol/L was 27.3%, 35.3%, and 19.4% for lorazepam, placebo, and no premedication, respectively (p = 0.356). The median (IQR) NAPCOMS pain score was 3.0 (0-6) for lorazepam, 4.4 (0-6) for placebo, and 3.4 (3-4) for no premedication (p = 0.428). With lorazepam, 75.9% of children reported experiencing a comfortable procedure, compared with 41.9% taking placebo and 34.5% with no premedication (p = 0.013). Transient tachycardia was the most frequent intraoperative adverse event, particularly with lorazepam (62.5%, p = 0.029). CONCLUSIONS: Oral lorazepam had no effect on patients' preoperative stress, as measured by salivary cortisol, but was associated with a higher rate of comfortable procedures. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov, Identifier NCT03180632.

Regulation of platelet-activating factor-mediated interleukin-6 promoter activation by the 48 kDa but not the 45 kDa isoform of protein tyrosine phosphatase non-receptor type 2.

BACKGROUND: An underlying state of inflammation is thought to be an important cause of cardiovascular disease. Among cells involved in the early steps of atherosclerosis, monocyte-derived dendritic cells (Mo-DCs) respond to inflammatory stimuli, including platelet-activating factor (PAF), by the induction of various cytokines, such as interleukin 6 (IL-6). PAF is a potent phospholipid mediator involved in both the onset and progression of atherosclerosis. It mediates its effects by binding to its cognate G-protein coupled receptor, PAFR. Activation of PAFR-induced signaling pathways is tightly coordinated to ensure specific cell responses. RESULTS: Here, we report that PAF stimulated the phosphatase activity of both the 45 and 48 kDa isoforms of the protein tyrosine phosphatase non-receptor type 2 (PTPN2). However, we found that only the 48 kDa PTPN2 isoform has a role in PAFR-induced signal transduction, leading to activation of the IL-6 promoter. In luciferase reporter assays, expression of the 48 kDa, but not the 45 kDa, PTPN2 isoform increased human IL-6 (hIL-6) promoter activity by 40% after PAF stimulation of HEK-293 cells, stably transfected with PAFR (HEK-PAFR). Our results suggest that the differential localization of the PTPN2 isoforms and the differences in PAF-induced phosphatase activation may contribute to the divergent modulation of PAF-induced IL-6 promoter activation. The involvement of PTPN2 in PAF-induced IL-6 expression was confirmed in immature Mo-DCs (iMo-DCs), using siRNAs targeting the two isoforms of PTPN2, where siRNAs against the 48 kDa PTPN2 significantly inhibited PAF-stimulated IL-6 mRNA expression. Pharmacological inhibition of several signaling pathways suggested a role for PTPN2 in early signaling events. Results obtained by Western blot confirmed that PTPN2 increased the activation of the PI3K/Akt pathway via the modulation of protein kinase D (PKD) activity. WT PKD expression counteracted the effect of PTPN2 on PAF-induced IL-6 promoter transactivation and phosphorylation of Akt. Using siRNAs targeting the individual isoforms of PTPN2, we confirmed that these pathways were also active in iMo-DCs. CONCLUSION: Taken together, our data suggest that PTPN2, in an isoform-specific manner, could be involved in the positive regulation of PI3K/Akt activation, via the modulation of PKD activity, allowing for the maximal induction of PAF-stimulated IL-6 mRNA expression.

Genomic Investigation of the Emergence of Invasive Multidrug-Resistant Salmonella enterica Serovar Dublin in Humans and Animals in Canada.

Salmonella enterica subsp. enterica serovar Dublin is a zoonotic pathogen that often leads to invasive bloodstream infections in humans that are multidrug resistant. Described here are the results of Canadian national surveillance of S Dublin from 2003 to 2015 in humans and bovines, principally collected through the Canadian Integrated Program for Antibiotic Resistance Surveillance (CIPARS). An increase in human infections due to multidrug-resistant (MDR) S Dublin was observed in 2010, many of which were bloodstream infections. Phylogenomic analysis of human and bovine isolates revealed a closely related network that differed by only 0 to 17 single nucleotide variants (SNVs), suggesting some potential transmission between humans and bovines. Phylogenomic comparison of global publicly available sequences of S Dublin showed that Canadian isolates clustered closely with those from the United States. A high correlation between phenotypic and genotypic antimicrobial susceptibility was observed in Canadian isolates. IS26 replication was widespread among U.S. and Canadian isolates and caused the truncation and inactivation of the resistance genes strA and blaTEM-1B A hybrid virulence and MDR plasmid (pN13-01125) isolated from a Canadian S Dublin isolate was searched against NCBI SRA data of bacteria. The pN13-01125 coding sequences were found in 13 Salmonella serovars, but S Dublin appears to be a specific reservoir. In summary, we have observed the rise of invasive MDR S Dublin in humans in Canada and found that they are closely related to bovine isolates and to American isolates in their mobile and chromosomal contents.

Measuring GPCR-Induced Activation of Protein Tyrosine Phosphatases (PTP) Using In-Gel and Colorimetric PTP Assays.

Given the increasing amount of data showing the importance of protein tyrosine phosphatases (PTPs) in G protein-coupled receptor (GPCR) signaling pathways, the modulation of this enzyme family by that type of receptor can become an important experimental question. Here, we describe two different methods, an in-gel and a colorimetric PTP assay, to evaluate the modulation of PTP activity after stimulation with GPCR agonists.

Age Dependent Dysfunction of Mitochondrial and ROS Metabolism Induced by Mitonuclear Mismatch.

Mitochondrial and nuclear genomes have to coevolve to ensure the proper functioning of the different mitochondrial complexes that are assembled from peptides encoded by both genomes. Mismatch between these genomes is believed to be strongly selected against due to the consequent impairments of mitochondrial functions and induction of oxidative stress. Here, we used a Drosophila model harboring an incompatibility between a mitochondrial tRNAtyr and its nuclear-encoded mitochondrial tyrosine synthetase to assess the cellular mechanisms affected by this incompatibility and to test the relative contribution of mitonuclear interactions and aging on the expression of impaired phenotypes. Our results show that the mitochondrial tRNA mutation caused a decrease in mitochondrial oxygen consumption in the incompatible nuclear background but no effect with the compatible nuclear background. Mitochondrial DNA copy number increased in the incompatible genotype but that increase failed to rescue mitochondrial functions. The flies harboring mismatch between nuclear and mitochondrial genomes had almost three times the relative mtDNA copy number and fifty percent higher rate of hydrogen peroxide production compared to other genome combinations at 25 days of age. We also found that aging exacerbated the mitochondrial dysfunctions. Our results reveal the tight interactions linking mitonuclear mismatch to mitochondrial dysfunction, mitochondrial DNA regulation, ROS production and aging.

Regulation of platelet-activating factor-induced interleukin-8 expression by protein tyrosine phosphatase 1B.

BACKGROUND: Platelet-activating factor (PAF) is a potent lipid mediator whose involvement in the onset and progression of atherosclerosis is mediated by, among others, the modulation of cytokine expression patterns. The presence of multiple potential protein-tyrosine phosphatase (PTP) 1B substrates in PAF receptor signaling pathways brought us to investigate its involvement in PAF-induced cytokine expression in monocyte-derived dendritic cells (Mo-DCs) and to study the pathways involved in this modulation. METHODS: We used in-vitro-matured human dendritic cells and the HEK-293 cell line in our studies. PTP1B inhibition was though siRNAs and a selective inhibitor. Cytokine expression was studied with RT-PCR, luciferase assays and ELISA. Phosphorylation status of kinases and transcription factors was studied with western blotting. RESULTS: Here, we report that PTP1B was involved in the modulation of cytokine expression in PAF-stimulated Mo-DCs. A study of the down-regulation of PAF-induced IL-8 expression, by PTP1B, showed modulation of PAF-induced transactivation of the IL-8 promoter which was dependent on the presence of the C/EBPß -binding site. Results also suggested that PTP1B decreased PAF-induced IL-8 production by a glycogen synthase kinase (GSK)-3-dependent pathway via activation of the Src family kinases (SFK). These kinases activated an unidentified pathway at early stimulation times and the PI3K/Akt signaling pathway in a later phase. This change in GSK-3 activity decreased the C/EBPß phosphorylation levels of the threonine 235, a residue whose phosphorylation is known to increase C/EBPß transactivation potential, and consequently modified IL-8 expression. CONCLUSION: The negative regulation of GSK-3 activity by PTP1B and the consequent decrease in phosphorylation of the C/EBPß transactivation domain could be an important negative feedback loop by which cells control their cytokine production after PAF stimulation.

Estimating diagnostic accuracy of fecal culture in liquid media for the detection of Mycobacterium avium subsp. paratuberculosis infections in Québec dairy cows: A latent class model.

A latent class model fit within a Bayesian framework was used to estimate the sensitivity and specificity of individual fecal culture (IFC) in liquid medium (Para TB culture liquid medium and BACTEC MGIT 960 system) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in Québec dairy cows. As a secondary objective, the within-herd paratuberculosis prevalence was estimated. A dataset including 21 commercial Québec dairy herds participating in previous research projects was retrospectively analyzed. In total, 1386 adult cows on which both IFC and serum-ELISA were available were included. The selected latent class model assumed conditional dependence between the tests. Non-informative priors for IFC accuracy and paratuberculosis prevalence were used while informative priors, obtained from the literature, were used for serum-ELISA accuracy. The WinBUGS statistical freeware was used to obtain posterior estimates (medians and 95% Bayesian credibility intervals (95% BCI)) for each parameter. The sensitivity and specificity estimates for IFC were 34.4% (95% BCI: 20.3-66.1) and 99.5% (95% BCI: 98.6-100), respectively. Sensitivity and specificity for serum-ELISA were 27.3% (95% BCI: 18.1-38.3) and 97.4% (95% BCI: 96.6-98.0). Median paratuberculosis within herd prevalence was estimated to be 0.3% (0-3.3). In conclusion, a higher sensitivity of IFC compared to serum-ELISA was observed both in the unconditional and conditional dependent models. Since the sensitivity of both IFC and serum-ELISA was relatively low, conditional dependence between the tests is more likely in the true disease positive animals. We hypothesize that conditional dependence arises because an unmeasured covariate influences the performance of both tests among disease positive animals causing both tests to incorrectly misclassify the animal as negative. One limitation of this study was the very low within herd prevalence of the participant herds.