Isolated discrete upper septal thickening in a non-referral cat population of senior and young cats.

INTRODUCTION/OBJECTIVES: Discrete upper septal thickening (DUST) is a phenotype of elderly people. The cardiac phenotype in senior cats has been incompletely described. We aimed to characterize the echocardiographic phenotype of senior cats, specifically to determine prevalence of DUST and hypertrophic cardiomyopathy (HCM). ANIMALS: One hundred and forty-nine healthy, normotensive cats. MATERIALS AND METHODS: Prospective cross-sectional study. Senior (≥9 years) and young (<6 years) cats were recruited from non-referral population. We defined DUST as an isolated basilar septal bulge, and HCM as left ventricular wall thickness ≥6 mm. An interventricular septum ratio (basal-to-mid septal thickness ratio) was calculated. We assessed for associations between clinical and echocardiographic variables and DUST. Data are presented as mean (±SD), median (range), or frequency (percentage). RESULTS: One-hundred and two senior and 47 young cats were enrolled. Aortoseptal angle (AoSA) was steeper in senior cats (137° (±14.5) vs. 145° (±12.3) in young cats, P=0.002). Eighteen cats had DUST (18/149, 12%), fourteen senior, and four young cats (P=0.4). Cats with DUST had steeper AoSA (125° (±8.3) vs. 142° (±13.7), P<0.0001) and higher interventricular septum ratio (1.4 (1.2-2.0) vs. 1.0 (0.7-1.8)). Univariable analysis showed decreased odds of DUST with greater AoSA (OR 0.9, P<0.0001), age was not associated with DUST. Twenty-nine senior cats had HCM (28.4%). DISCUSSION/CONCLUSIONS: Prevalence of DUST was 12%. There was no association between age and DUST. Smaller/steeper AoSA was the main factor associated with DUST. There was a high prevalence of HCM in this senior population.

Prevalence of Trauma-Induced Hypocalcemia in the Prehospital Setting.

BACKGROUND: Recent data published by the Special Operations community suggest the Lethal Triad of Trauma should be changed to the Lethal Diamond, to include coagulopathy, acidosis, hypothermia, and hypocalcemia. The purpose of this study is to determine the prevalence of trauma-induced hypocalcemia in level I and II trauma patients. METHODS: This is a retrospective cohort study conducted at a level I trauma center and Special Operations Combat Medic (SOCM) training site. Adult patients were identified via trauma services registry from September 2021 to April 2022. Patients who received blood products prior to emergency department (ED) arrival were excluded from the study. Ionized calcium levels were utilized in this study. RESULTS: Of the 408 patients screened, 370 were included in the final analysis of this cohort. Hypocalcemia was noted in 189 (51%) patients, with severe hypocalcemia identified in two (<1%) patients. Thirty-two (11.2%) patients had elevated international normalized ratio (INR), 34 (23%) patients had pH <7.36, 21 (8%) patients had elevated lactic acid, and 9 (2.5%) patients had a temperature of <35°C. CONCLUSION: Hypocalcemia was prevalent in half of the trauma patients in this cohort. The administration of a calcium supplement empirically in trauma patients from the prehospital environment and prior to blood transfusion is not recommended until further data prove it beneficial.

Spontaneous Ruptured Pyomyoma in a Nulligravid Female: A Case Report and Review of the Literature.

INTRODUCTION: Pyomyoma, or suppurative leiomyoma, is a rare complication of uterine fibroids. It occurs most commonly in the setting of pregnancy, the immediate postpartum period, or postmenopausal status. It may also arise after recent uterine instrumentation, after uterine artery embolization, or in immunocompromised patients. The most likely cause of pyomyoma is vascular compromise followed by bacterial seeding from direct, hematogenous, or lymphatic spread. Diagnosis is difficult, as the condition is rare, presents with vague symptoms, and is difficult to identify on imaging. Definitive diagnosis is only possible with surgery. Pathology shows a degenerating fibroid with hemorrhage, necrosis, cystic degeneration, and/or inflammatory change. Cultures of the pus contained within often show polymicrobial infection. CASE PRESENTATION: Our patient is a 24-year-old nulligravid female who presented with a surgical abdomen, fever, hypotension, and leukocytosis. She had no significant prior medical or surgical history, no history of uterine instrumentation, and no history of pelvic infection; she was not currently sexually active at the time of presentation. She was taken to the operating room, where she underwent diagnostic laparoscopy. This showed a ruptured pyomyoma originating in the left broad ligament. She then underwent laparoscopic myomectomy. She was transferred to the ICU intubated; she slowly recovered on IV antibiotics and was discharged home on postoperative day 10. DISCUSSION: Pyomyoma is a rare condition and is even rarer in premenopausal patients without recent history of pregnancy or uterine instrumentation. This demonstrates an unusual case of spontaneous pyomyoma in the absence of risk factors, other than a history of known fibroids. Pyomyoma should be considered as a diagnosis in patients with sepsis, history of fibroids, and no other identifiable source of infection.

Spatial clusters of latent tuberculous infection, Connecticut, 2010-2014.

SETTING: In the United States, tuberculosis (TB) control is increasingly focusing on the identification of persons with latent tuberculous infection (LTBI). OBJECTIVE: To characterize the local epidemiology of LTBI in Connecticut, USA. METHODS: We used spatial analyses 1) to identify census tract-level clusters of reported LTBI and TB disease in Connecticut, 2) to compare persons and populations in clusters with those not in clusters, and 3) to compare persons with LTBI to those with TB disease. RESULTS: Significant census tract-level spatial clusters of LTBI and TB disease were identified. Compared with persons with LTBI in non-clustered census tracts, those in clustered census tracts were more likely to be foreign-born and less likely to be of white non-Hispanic ethnicity. Populations in census tract clusters of high LTBI prevalence had greater crowding, persons living in poverty, and persons lacking health care insurance than populations not in clustered census tracts. Persons with LTBI were less likely than those with TB disease to be of Asian ethnicity, and persons with LTBI were more likely than those with TB disease to reside in a clustered census tract. CONCLUSIONS: Characterizing fine-scale populations at risk for LTBI supports effective and culturally accessible screening and treatment programs.

mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells.

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCDcl1 parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCDcl1 cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCDcl1 cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCDcl1 cells being precursor cells.

Precise spatio-temporal control of rapid optogenetic cell ablation with mem-KillerRed in Zebrafish.

The ability to kill individual or groups of cells in vivo is important for studying cellular processes and their physiological function. Cell-specific genetically encoded photosensitizing proteins, such as KillerRed, permit spatiotemporal optogenetic ablation with low-power laser light. We report dramatically improved resolution and speed of cell targeting in the zebrafish kidney through the use of a selective plane illumination microscope (SPIM). Furthermore, through the novel incorporation of a Bessel beam into the SPIM imaging arm, we were able to improve on targeting speed and precision. The low diffraction of the Bessel beam coupled with the ability to tightly focus it through a high NA lens allowed precise, rapid targeting of subsets of cells at anatomical depth in live, developing zebrafish kidneys. We demonstrate that these specific targeting strategies significantly increase the speed of optoablation as well as fish survival.

A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial.

INTRODUCTION: The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. MATERIALS AND METHODS: A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. RESULTS: There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p=0.045) and Nef (p=0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p<0.05) in Gag (n=10), Pol (n=7) and Nef (n=10), although they did not remain significant after adjustment for multiple comparisons. We found the epitope sieve effect in Step was driven by HLA A∗02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. DISCUSSION: This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes.

Early-life perturbations in glucocorticoid activity impacts on the structure, function and molecular composition of the adult zebrafish (Danio rerio) heart.

BACKGROUND: Transient early-life perturbations in glucocorticoids (GC) are linked with cardiovascular disease risk in later life. Here the impact of early life manipulations of GC on adult heart structure, function and gene expression were assessed. METHODS AND RESULTS: Zebrafish embryos were incubated in dexamethasone (Dex) or injected with targeted glucocorticoid receptor (GR) morpholino knockdown (GR Mo) over the first 120 h post fertilisation (hpf); surviving embryos (>90%) were maintained until adulthood under normal conditions. Cardiac function, heart histology and cardiac genes were assessed in embryonic (120 hpf) and adult (120 days post fertilisation (dpf)) hearts. GR Mo embryos (120 hpf) had smaller hearts with fewer cardiomyocytes, less mature striation pattern, reduced cardiac function and reduced levels of vmhc and igf mRNA compared with controls. GR Mo adult hearts were smaller with diminished trabecular network pattern, reduced expression of vmhc and altered echocardiographic Doppler flow compared to controls. Dex embryos had larger hearts at 120 hpf (Dex 107.2 ± 3.1 vs. controls 90.2 ± 1.1 µm, p < 0.001) with a more mature trabecular network and larger cardiomyocytes (1.62 ± 0.13 cells/µm vs control 2.18 ± 0.13 cells/µm, p < 0.05) and enhanced cardiac performance compared to controls. Adult hearts were larger (1.02 ± 0.07 µg/mg vs controls 0.63 ± 0.06 µg/mg, p = 0.0007), had increased vmhc and gr mRNA levels. CONCLUSION: Perturbations in GR activity during embryonic development results in short and long-term alterations in the heart.

Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio).

While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11ß-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research.

Renal pericytes: multifunctional cells of the kidneys.

Pericytes have become a hot topic in renal biology. They play a critical physiological role in vessel development, maintenance and remodelling through active communication with their vascular partners-endothelial cells-and modulation of extracellular matrix proteins. Multiple functions for renal pericytes have been described; specialised perivascular populations participate in glomerular filtration, regulate medullary blood flow and contribute to kidney fibrosis by differentiation into collagen-generating myofibroblasts. Interestingly, the origin of renin-producing cells of the juxtaglomerular region is attributed to the perivascular cell lineage; we have observed the coincidence of renin and pericyte marker expression during human kidney development. Finally, pericytes have been shown to share features with mesenchymal stem cells, which places them as potential renal progenitor cell candidates. Since renal diseases are often associated with microvascular complications, renal pericytes may emerge as new targets for the treatment of kidney disease.

Proteomic analysis of epidermal mucus from sea lice-infected Atlantic salmon, Salmo salar L.

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds.

Micro-endoscope for in vivo widefield high spatial resolution fluorescent imaging.

In this paper we report the design, testing and use of a scannerless probe specifically for minimally invasive imaging of deep tissue in vivo with an epi-fluorescence modality. The probe images a 500 µm diameter field of view through a 710 µm outer diameter probe with a maximum tissue penetration depth of 15 mm specifically configured for eGFP imaging. Example results are given from imaging the pituitary gland of rats and zebrafish hearts with lateral resolution of 2.5 µm.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

Expression, purification and use of the soluble domain of Lactobacillus paracasei beta-fructosidase to optimise production of bioethanol from grass fructans.

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2.

BACKGROUND: Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. METHODS: We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, > or = 250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. RESULTS: A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P=0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log(10) copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2-positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir were observed. CONCLUSIONS: Daily acyclovir therapy did not reduce the risk of transmission of HIV-1, despite a reduction in plasma HIV-1 RNA of 0.25 log(10) copies per milliliter and a 73% reduction in the occurrence of genital ulcers due to HSV-2. (ClinicalTrials.gov number, NCT00194519.)

Conjugation of fluorochromes to antibodies.

Immunolocalization of antigen via fluorescence requires that fluorochromes be linked either to the primary antibody (direct method) or to a second antibody (indirect method) to provide a fluorescent signal to mark the site of antibody-antigen binding. Of these two methods, the indirect technique is generally more useful and practical. Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules. Provided are step-by-step instructions for conjugating isothiocyanate derivates of fluorescein and sulfonyl chloride derivatives of rhodamine to the amine groups of antibodies.

Fluorochromes: properties and characteristics.

Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, and how well that image can be distinguished from background fluorescence or interfering fluorescence signals. A wide variety of fluorochromes are available, with emitting wavelengths that range from the blue-violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their extinction coefficients, quantum yields, susceptibility to photobleaching, the wavelengths at which they maximally absorb excitatory and emit fluorescent light, and how far apart those wavelength maxima are separated. Additional choices for fluorescent labeling of antibodies are provided by the availability of fluorescent quantum dots. Informed choices of fluorochromes can obviate many problems, particularly with regard to situations in which two or more antigens are to be localized simultaneously within a specimen.

Overview of conventional fluorescence photomicrography.

In an age of digital imaging, photographic film still provides a viable and effective means for recording fluorescence images by photomicrography. To maximize the quality of results that are obtained, a photographic emulsion with sufficient sensitivity for the low light level characteristic of Immunofluorescence must be selected, exposures adjusted for reciprocity failure, and modern, high numerical aperture objective lenses employed to produce the brightest possible image. Mounting media that reduce the effects of photobleaching on fluorochromes also help to maintain image brightness, and so reduce exposure times. Digital scanning of film-based micrographs provides the convenience of utilizing image processing software to adjust image density and contrast, and to produce quality prints.