Identification of promoter activity in gene-less cassettes from Vibrionaceae superintegrons.

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

"It's Like A Partnership": Exploring the Primary Care Experiences and Patient-Defined Goals of People Who Use Drugs.

BACKGROUND: Primary care is an important yet underutilized resource in addressing the overdose crisis. Previous studies have identified important aspects of primary care for people who use drugs (PWUD) and have found patient involvement in healthcare decisions and goal-setting to be especially critical. However, there has been limited research describing the primary care goals of PWUD. In harm reduction settings, where it is imperative that PWUD set their own goals, this research gap becomes especially relevant. OBJECTIVE: To explore how PWUD navigate primary care with a focus on understanding their primary care goals. DESIGN: A qualitative study using semi-structured interviews. PARTICIPANTS: PWUD currently engaged in primary care at the Respectful and Equitable Access to Comprehensive Healthcare (REACH) Program, a harm reduction-based primary care program in New York City. APPROACH: Between June 2022 and August 2022, we conducted 17 semi-structured interviews. Informed by phenomenology, transcripts were coded using both inductive and deductive codes and themes were developed using thematic analysis approaches. KEY RESULTS: Phenomenological analysis identified four core components that, together, created an experience that participants described as "a partnership" between patient and provider: (1) patient-provider collaboration around patient-defined healthcare goals; (2) support provided by harm reduction-based approaches to primary care anchored in incrementalism and flexibility; (3) care teams' ability to address healthcare system fragmentation; and (4) the creation of social connections through primary care. This holistic partnership fostered positive primary care experiences and supported participants' self-defined care goals, thereby facilitating meaningful care outcomes. CONCLUSIONS: To best meet the primary care goals of PWUD, these findings underscore the importance of primary care providers and programs facilitating such partnerships through organizational-level support anchored in harm reduction. Future research should explore how these experiences in primary care affect patient health outcomes, ultimately shaping best practices in the provision of high-quality primary care for PWUD.

The expression of aminoglycoside resistance genes in integron cassettes is not controlled by riboswitches.

Regulation of gene expression is a key factor influencing the success of antimicrobial resistance determinants. A variety of determinants conferring resistance against aminoglycosides (Ag) are commonly found in clinically relevant bacteria, but whether their expression is regulated or not is controversial. The expression of several Ag resistance genes has been reported to be controlled by a riboswitch mechanism encoded in a conserved sequence. Yet this sequence corresponds to the integration site of an integron, a genetic platform that recruits genes of different functions, making the presence of such a riboswitch counterintuitive. We provide, for the first time, experimental evidence against the existence of such Ag-sensing riboswitch. We first tried to reproduce the induction of the well characterized aacA5 gene using its native genetic environment, but were unsuccessful. We then broadened our approach and analyzed the inducibility of all AgR genes encoded in integrons against a variety of antibiotics. We could not observe biologically relevant induction rates for any gene in the presence of several aminoglycosides. Instead, unrelated antibiotics produced mild but consistently higher increases in expression, that were the result of pleiotropic effects. Our findings rule out the riboswitch control of aminoglycoside resistance genes in integrons.

The value of repeat kidney biopsy during an atypical course of membranous nephropathy.

BACKGROUND: The clinical trajectory for patients with primary membranous nephropathy ranges widely from spontaneous remission to a rapid decline in kidney function. Etiologies for rapid progression with membranous nephropathy include concurrent bilateral renal vein thrombosis, malignant hypertension, and crescentic membranous nephropathy. Given the wide heterogeneity in prognosis, timing of immunosuppressive therapy is often challenging and centers around an individual patient's perceived risk for rapidly progressive disease. CASE PRESENTATION: Herein, we describe the clinical course of a young patient who initially developed a typical presentation of membranous nephropathy with consistent kidney biopsy findings. Given clinical stability, a six month observation period was undertaken prior to initiating immunosuppression. Within this observation window, the patient developed community acquired pneumonia followed several weeks later by a sudden, rapid decline in kidney function requiring dialysis. Repeat kidney biopsy revealed post-infectious glomerulonephritis superimposed upon a background of membranous nephropathy. Immunosuppressive therapy resulted in a favorable long-term outcome with normalization of kidney function and remission of nephrotic syndrome. To our knowledge, this is the first report of the simultaneous occurrence of these two glomerular disease processes. CONCLUSION: This case illustrates the value of repeat kidney biopsy during an atypical course of membranous nephropathy. Superimposed glomerular disease processes should be considered during a course of rapidly progressive membranous nephropathy.

Doppler ultrasonographic evaluation of medically treated female dogs with cystic endometrial hyperplasia-pyometra complex.

Monitoring the treatment outcome of canine cystic endometrial hyperplasia-pyometra complex (CEH-P) is essential to identify nonresponding patients who might require a rapid intervention to avoid life-threatening conditions. Uterine artery Doppler characterization may contribute to monitoring medically treated CEH-P, but published studies are currently lacking. Therefore, the aim of this longitudinal prospective observational study was to evaluate uterine artery resistance changes in the medical treatment of female dogs with CEH-P. Twelve diestrous female dogs suffering from CEH-P were treated with a combined protocol of cabergoline, cloprostenol, and antibiotics. The animals were clinically and ultrasonographically evaluated before the beginning of treatment (day 0) and on days 3, 7, 14, 21, and 28. The widest transverse diameter and luminal diameter of uterine horns were measured, as well as the peak systolic velocity (PSV) and end diastolic velocity (EDV) of uterine arteries. The resistance index (RI), pulsatility index (PI), and notch deep index (NDI) were calculated. On day 3, nine of 12 animals showed clinical improvement. In all these female dogs, the widest transverse sectional diameter (P < 0.01), luminal diameter (P < 0.01), PSV (P < 0.01), and EDV (P < 0.01) progressively decreased, while RI (P < 0.01), PI (P < 0.01), and NDI (P < 0.01) increased up to day 21. In nonresponding dogs (3/12), Doppler parameters remained unchanged. Although comparisons of PSV, EDV, and NDI were significantly different, these results should be cautiously interpreted due to the low statistical power. female dogs that responded to this treatment showed an increase in uterine artery resistance, along with clinical and ultrasonographic improvement.

Transitioning Clinical Care for People Who Use Drugs to Telemedicine: Lessons Learned One Year into the COVID-19 Pandemic.

Background: As a harm reduction-focused primary care clinic for people who use drugs, the Respectful and Equitable Access to Comprehensive Healthcare (REACH) Program faced multiple barriers due to the COVID-19 pandemic. We describe and evaluate how the telemedicine-driven adaptations REACH made allowed the program to engage its patients. Methods: REACH expanded its telemedicine capabilities by transitioning its in-person clinic and methods of connecting with referrals to telemedicine. The program provided patients with phones to increase access to needed technology. Results: Throughout 2020, patient visits continuously shifted from being entirely in-person, to entirely telemedicine, to a hybrid model. Clinic show rates averaged 71% with this hybrid model, compared with 57% pre-COVID-19. Phones were distributed to 88 patients, 77% of which engaged in at least one telemedicine visit. Conclusions: Telemedicine allowed REACH to provide uninterrupted care during the pandemic. The program is now refining its hybrid model of telemedicine and in-person care to more equitably serve all patients.

Cardiac up-regulation of NBCe1 emerges as a beneficial consequence of voluntary wheel running in mice.

Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, µm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice. These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.

Efficacy of the Parasympathetic Tone Activity monitor to assess nociception in healthy dogs anaesthetized with propofol and sevoflurane.

OBJECTIVE: To compare a Parasympathetic Tone Activity (PTA) monitor with cardiovascular changes in invasive mean arterial pressure (IMAP) and heart rate (HR) when evaluating the response to nociceptive stimuli in anaesthetized dogs. STUDY DESIGN: Prospective experimental study. ANIMALS: A group of nine (seven male and two female) adult Beagle dogs weighing 13.4 ± 1.5 kg (mean ± standard deviation). METHODS: Anaesthesia was induced with propofol and maintained with sevoflurane in oxygen. Electrical stimuli of different nociceptive intensities were applied for 30 seconds. Stimuli were classified in each patient according to the response obtained (relevant change ≥ 20%) as low (no response), medium (PTA only) or high (PTA and IMAP/HR). Immediate and averaged values of PTA, IMAP and HR were recorded every second from 60 seconds before to 120 seconds after application of the nociceptive stimulus. Time to nociceptive response and peak response were evaluated with analysis of variance and t test. RESULTS: Immediate PTA baseline values did not differ significantly before application of the low, medium and high stimuli (73 ± 15, p = 0.966). Immediate PTA response was observed with the medium stimulus at 33 ± 7 seconds with a maximum decrease of 57 ± 13% at 69 ± 5 seconds. With the high stimulus, the immediate PTA response was of a similar magnitude to the medium stimulus with a response at 28 ± 7 seconds (p = 0.221) and a maximum decrease of 68 ± 15% (p = 0.115) at 72 ± 7 seconds (p = 0.436). The cardiovascular change occurred (22 ± 8 seconds) prior to the immediate PTA response (p = 0.032). CONCLUSIONS AND CLINICAL RELEVANCE: The PTA monitor detected nociceptive stimuli at lower intensities than those eliciting cardiovascular changes. However, nociceptive stimuli of higher intensities provoked cardiovascular changes that occurred before a PTA response was observed.

Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob-/- type 2 diabetic mice.

Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob-/-) and control heterozygous littermates (ob+/-). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob-/- mice at 9 and 16 weeks. ob-/- mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob-/-vs. ob+/- mice, and augmented Ser703 phosphorylation on NHE1 in ob-/- hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob-/- mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob-/- mice vs. ob+/-. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob-/- mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.

Novel Inducers of the Expression of Multidrug Efflux Pumps That Trigger Pseudomonas aeruginosa Transient Antibiotic Resistance.

The study of the acquisition of antibiotic resistance (AR) has mainly focused on inherited processes, namely, mutations and acquisition of AR genes. However, inducible, noninheritable AR has received less attention, and most information in this field derives from the study of antibiotics as inducers of their associated resistance mechanisms. Less is known about nonantibiotic compounds or situations that can induce AR during infection. Multidrug resistance efflux pumps are a category of AR determinants characterized by the tight regulation of their expression. Their contribution to acquired AR relies in their overexpression. Here, we analyzed potential inducers of the expression of the chromosomally encoded Pseudomonas aeruginosa clinically relevant efflux pumps, MexCD-OprJ and MexAB-OprM. For this purpose, we developed a set of luxCDABE-based P. aeruginosa biosensor strains, which allows the high-throughput analysis of compounds able to modify the expression of these efflux pumps. Using these strains, we analyzed a set of 240 compounds present in Biolog phenotype microarrays. Several inducers of the expression of the genes that encode these efflux pumps were found. The study focused in dequalinium chloride, procaine, and atropine, compounds that can be found in clinical settings. Using real-time PCR, we confirmed that these compounds indeed induce the expression of the mexCD-oprJ operon. In addition, P. aeruginosa presents lower susceptibility to ciprofloxacin (a MexCD-OprJ substrate) when dequalinium chloride, procaine, or atropine are present. This study emphasizes the need to study compounds that can trigger transient AR during antibiotic treatment, a phenotype difficult to discover using classical susceptibility tests.

Analysis of the Pseudomonas aeruginosa Aminoglycoside Differential Resistomes Allows Defining Genes Simultaneously Involved in Intrinsic Antibiotic Resistance and Virulence.

High-throughput screening of transposon insertion libraries is a useful strategy for unveiling bacterial genes whose inactivation results in an altered susceptibility to antibiotics. A potential drawback of these studies is they are usually based on just one model antibiotic for each structural family, under the assumption that the results can be extrapolated to all members of said family. To determine if this simplification is appropriate, we have analyzed the susceptibility of mutants of Pseudomonas aeruginosa to four aminoglycosides. Our results indicate that each mutation produces different effects on susceptibility to the tested aminoglycosides, with only two mutants showing similar changes in the susceptibility to all studied aminoglycosides. This indicates that the role of a particular gene in the resistome of a given antibiotic should not be generalized to other members of the same structural family. Five aminoglycoside-hypersusceptible mutants inactivating glnD, hflK, PA2798, PA3016, and hpf were chosen for further analysis in order to elucidate if lower aminoglycoside susceptibility correlates with cross-hypersusceptibility to other antibiotics and with impaired virulence. Our results indicate that glnD inactivation leads to increased cross-susceptibility to different antibiotics. The mutant in this gene is strongly impaired in virulence traits such as pyocyanin production, biofilm formation, elastase activity, and swarming motility and the ability to kill Caenorhabditis elegans Thus, GlnD might be an interesting target for developing antibiotic coadjuvants with antiresistance and antivirulence properties against P. aeruginosa.

Mechanisms and phenotypic consequences of acquisition of tigecycline resistance by Stenotrophomonas maltophilia.

OBJECTIVES: To elucidate the potential mutation-driven mechanisms involved in the acquisition of tigecycline resistance by the opportunistic pathogen Stenotrophomonas maltophilia. The mutational trajectories and their effects on bacterial fitness, as well as cross-resistance and/or collateral susceptibility to other antibiotics, were also addressed. METHODS: S. maltophilia populations were submitted to experimental evolution in the presence of increasing concentrations of tigecycline for 30 days. The genetic mechanisms involved in the acquisition of tigecycline resistance were determined by WGS. Resistance was evaluated by performing MIC assays. Fitness of the evolved populations and individual clones was assessed by measurement of the maximum growth rates. RESULTS: All the tigecycline-evolved populations attained high-level resistance to tigecycline following different mutational trajectories, yet with some common elements. Among the mechanisms involved in low susceptibility to tigecycline, mutations in the SmeDEF efflux pump negative regulator smeT, changes in proteins involved in the biogenesis of the ribosome and modifications in the LPS biosynthesis pathway seem to play a major role. Besides tigecycline resistance, the evolved populations presented cross-resistance to other antibiotics, such as aztreonam and quinolones, and they were hypersusceptible to fosfomycin, suggesting a possible combination treatment. Further, we found that the selected resistance mechanisms impose a relevant fitness cost when bacteria grow in the absence of antibiotic. CONCLUSIONS: Mutational resistance to tigecycline was easily selected during exposure to this antibiotic. However, the fitness cost may compromise the maintenance of S. maltophilia tigecycline-resistant populations in the absence of antibiotic.

Histopathological Study on the Proposed Pathogenesis of Intratarsal Keratinous Cysts.

PURPOSE: Intratarsal keratinous cysts (IKCs) are a recently described entity that is frequently misdiagnosed clinically as chalazia and mislabeled in the literature as "intratarsal epidermal inclusion cysts" or "epidermoid cysts." It is important to accurately diagnose IKCs and distinguish them from chalazia because IKCs require a complete surgical excision and can exhibit multiple recurrences following curettage. The authors performed a retrospective case series to further elucidate the pathogenesis of IKCs and to determine the diagnostically optimal panel of stains for diagnosis. METHODS: A study group of 8 specimens of IKCs and control specimens of epidermal inclusion cysts were obtained from their pathology laboratories. The authors compared the histological and immunohistochemical profile of IKCs and epidermal inclusion cysts by staining sections from each specimen with hematoxylin and eosin, periodic acid-Schiff, Masson trichrome, cytokeratin 5, cytokeratin 17, carcinoembryonic antigen, and epithelial membrane antigen. The immunoreactivity data were then analyzed using a 2-tailed Mann-Whitney test, assuming a nonparametric population (p < 0.05 is significant). RESULTS: Histopathologically, IKCs are embedded in the tarsus lined by stratified squamous epithelium with an inner undulating cuticle filled with a compact keratinous-appearing material. The authors demonstrate that IKCs develop progressively from dilated meibomian ducts to the formation of complete cysts with their markers. The most valuable immunochemical stains to diagnose IKC were cytokeratin 17, carcinoembryonic antigen, and epithelial membrane antigen (p < 0.05 with each). CONCLUSIONS: These findings provide a better understanding of the pathogenesis and the immunohistochemical findings of this relatively new entity allowing for more appropriate diagnosis of IKCs aiming to reduce future complications from their management.

Primary membranous nephropathy presenting with crescentic glomerulonephritis 25 years after initial presentation: A case report
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Membranous nephropathy (MN) is a common cause of nephrotic syndrome. Rarely, it can present with rapidly-progressive renal failure and hematuria. While this may be due to lupus nephritis, superimposed anti-glomerular basement membrane (GBM) disease, or antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, there have been rare reports of anti-GBM - and ANCA-negative crescentic glomerulonephritis presenting in primary membranous nephropathy (pMN). We present the case of a patient with long-standing pMN who developed acute deterioration of renal function and was found to have a flare of MN along with crescentic glomerulonephritis (GN) despite a negative serum ANCA, anti-GBM, and antinuclear antibody (ANA) work-up. He was started on dialysis and immunosuppressive therapy, and eventually recovered enough renal function to become dialysis-independent. A brief review of available literature suggests that crescentic GN presenting with pMN is a rare but established entity. Much more rarely has it been reported to occur in patients with previously-diagnosed pMN. In these contexts, crescentic GN may be occurring as the most severe manifestation of pMN rather than as a separate entity. Immunosuppressive therapy is often given, however, prognosis is guarded as half of patients will have worsening renal function and a quarter will develop end-stage renal disease.
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Sickle cell disease: a case report of renal amyloidosis.

BACKGROUND: The development of proteinuria and reduced glomerular filtration rate is associated with higher mortality among patients with sickle cell disease (SCD). AA amyloidosis, also associated with increased mortality, in SCD is rare. We present a case of a woman with homozygous sickle cell disease with nephrotic syndrome and antibodies to double stranded DNA without clinical features of systemic lupus erythematosus. Kidney biopsy reveals AA amyloidosis and is the first report of concomitant AA amyloidosis with antibodies to double stranded DNA in SCD. CASE PRESENTATION: A 40-year-old Central African woman with homozygous sickle cell disease and history of vaso-occlusive pain crises undergoes kidney biopsy for nephrotic-range proteinuria. Kidney biopsy reveals AA type amyloidosis, which is a rare manifestation of SCD in the kidney. Her anemia worsens with an ACE inhibitor, initiated to reduce proteinuria and limit GFR decline, so it was discontinued. Hydroxyurea, shown to decrease the frequency of vaso-occlusive crises and lower proteinuria, was subsequently initiated but then discontinued due to worsening anemia. Unfortunately, her glomerular filtration rate worsens. CONCLUSIONS: AA amyloidosis and antibodies to double stranded DNA can occur in sickle cell disease. ACE inhibition and hydroxyurea decrease proteinuria so they may limit progression of chronic kidney disease. Hydroxyurea also decreases frequency of vaso-occlusive pain crises so it might be helpful in limiting progression of renal AA amyloidosis. However, further studies are needed to determine optimal treatment strategies for AA amyloidosis in sickle cell disease.

Ultrasonographic evaluation of ovarian morphology and blood flow in prepubertal and pubertal cats.

The aim of this study was to evaluate two-dimensional and Doppler ultrasonographic changes of feline ovaries before and during puberty. Nine, 3-month-old female cats were followed until puberty (Day 1). Two-dimensional and Doppler ultrasound evaluations of the ovaries were carried out on Days -50, -20, -7 and 1. Longitudinal and transverse sections of the ovaries were measured and all anechoic spherical structures were considered to be follicles. The number of follicles >1 mm and the maximum diameter of the largest follicle were recorded. Peak systolic velocity and end diastolic velocity of intraovarian arteries were also measured to automatically calculate the resistive index (RI). The mean ovarian longitudinal diameter increased gradually throughout the study from 8.6 to 10.7 mm (p < 0.05). While four cats presented multiple anechoic spherical structures <1 mm diameter throughout the study, the remaining five animals had these structures only on Days -50 and -20. On Days -20, -7 and 1, the mean number of follicles were 1.4 ± 0.7, 2.5 ± 0.8 and 4.8 ± 1 respectively (p < 0.01). The largest follicles at the same time points were 1.1 ± 0.2 mm, 1.9 ± 0.3 mm and 2.6 ± 0.5 mm respectively (p < 0.05). The RI of the intraovarian arteries declined throughout the study period (p < 0.01). It is concluded that, in female cats, ovarian dimensions, follicle number and intraovarian blood flow increased from 3 months of age to puberty.

Uterine two-dimensional and Doppler ultrasonographic evaluation of feline pyometra.

The aim of this study was to compare uterine two-dimensional and Doppler ultrasonographic parameters in queens suffering from pyometra from those in early pregnancy. Secondly, the effect of the presence of clinical signs of systemic illness on these parameters was also described. Fourteen post-oestrous queens, with uterine luminal content in the absence of embryos were included. According to their outcome (pyometra surgery or parturition), the queens were retrospectively assigned to one of the following groups: Pyometra (PYO; n = 7) or pregnant (PRG; n = 7). In all the queens, two-dimensional and Doppler ultrasound examinations of the uterus were performed. The presence or absence of clinical signs of systemic illness was recorded. The widest cross-sectional diameter (UD), uterine wall thickness (WT), uterine lumen contents (LC) and uterine artery resistance index (RI) were measured. Uterine horn diameter was higher in PYO group than in PRG group (p < 0.05), while WT (p > 0.1) and LC (p = 0.09) did not differ between groups. Values of RI for PYO and PRG groups were 0.61 ± 0.03 vs 0.53 ± 0.09 (p < 0.05), respectively. PYO cats suffering from clinical signs of systemic illness showed larger UD than those without signs (p < 0.01). It is concluded that two-dimensional and Doppler ultrasound might be useful to distinguish queens suffering from pyometra from those in early pregnancy. Secondly, the clinical signs of systemic illness were associated with a larger UD.

Calcium-calmodulin-dependent protein kinase mediates the intracellular signalling pathways of cardiac apoptosis in mice with impaired glucose tolerance.

KEY POINTS: Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose-rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3 H]ryanodine binding and Ca2+ /calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2-S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD-WT mice, which was prevented by co-treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD-SR-autocamide inhibitory peptide (AIP) mice, expressing the SR-targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD-SR-AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD-WT mice but not in FRD-S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. ABSTRACT: The impact of cardiac apoptosis in pre-diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose-rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca2+ /calmodulin-protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca2+ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre-diabetes. We generated a pre-diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca2+ spontaneous events in the absence of SR Ca2+ load alterations vs. control-diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [3 H]ryanodine binding and CaMKII phosphorylation of RyR2-S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia-induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD-WT mice. Co-treatment with the reactive oxygen species scavenger Tempol prevented FRD-induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD-SR-AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR-mitochondrial distance vs. CD-WT hearts. This remodelling was prevented in AC3I mice, with cardiac-targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca2+ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre-diabetic heart. The FRD-induced decrease in SR-mitochondrial distance is likely to additionally favour Ca2+ transit between the two organelles.