Blocking mitochondrial Zn2+ accumulation after ischemia reduces mitochondrial dysfunction and neuronal injury.

Zn2+ is an important contributor to ischemic brain injury and recent studies support the hypothesis that mitochondria are key sites of its injurious effects. In murine hippocampal slices (both sexes) subjected to oxygen glucose deprivation (OGD), we found that Zn2+ accumulation and its entry into mitochondria precedes and contributes to the induction of acute neuronal death. In addition, if the ischemic episode is short (and sublethal), there is ongoing Zn2+ accumulation in CA1 mitochondria after OGD that may contribute to their delayed dysfunction. Using this slice model of sublethal OGD, we have now examined Zn2+ contributions to the progression of changes evoked by OGD and occurring over 4-5 hours. We detected progressive mitochondrial depolarization occurring from ∼ 2 hours after ischemia, a large increase in spontaneous synaptic activity between 2-3 hours, and mitochondrial swelling and fragmentation at 4 hours. Blockade of the primary route for Zn2+ entry, the mitochondrial Ca2+ uniporter (MCU; with ruthenium red, RR) or Zn2+ chelation shortly after OGD withdrawal substantially attenuated the mitochondrial depolarization and the changes in synaptic activity. RR also largely reversed the mitochondrial swelling. Finally, using an in vivo rat (male) asphyxial cardiac arrest (CA) model of transient global ischemia, we found that ∼8 min asphyxia induces considerable injury of CA1 neurons 4 hours later that is associated with strong Zn2+ accumulation within many damaged mitochondria. These effects were substantially attenuated by infusion of RR upon reperfusion. Our findings highlight mitochondrial Zn2+ accumulation after ischemia as a possible target for neuroprotective therapy.SIGNIFICANCE STATEMENT:Brain ischemia is a leading cause of mortality and long-term disability that still lacks effective treatment. After transient ischemia delayed death of neurons occurs in vulnerable brain regions. There is a critical need to understand mechanisms of this delayed neurodegeneration which can be targeted for neuroprotection. We found progressive and long-lasting mitochondrial Zn2+ accumulation to occur in highly vulnerable CA1 neurons after ischemia. Here we demonstrate that this Zn2+ accumulation contributes strongly to deleterious events occurring after ischemia including mitochondrial dysfunction, swelling and structural changes. We suggest that this mitochondrial Zn2+ entry may constitute a promising target for development of therapeutic interventions to be delivered after termination of an episode of transient global ischemia.

Laparoscopic transanal minimally invasive surgery (L-TAMIS) versus robotic TAMIS (R-TAMIS): short-term outcomes and costs of a comparative study.

BACKGROUND: Transanal minimally invasive surgery (TAMIS) has gained worldwide popularity as a method for the local excision of rectal neoplasms. However, it is technically demanding due to limited working space. Robotic TAMIS offers potential enhanced dexterity and ability while allowing for a more aggressive resection with a stable platform. The objective of this study was to review a single institution experience between laparoscopic (L-TAMIS) and robotic TAMIS (R-TAMIS) for treatment of rectal neoplasms and determine if there are significant differences on outcomes. METHODS: Forty consecutive patients with rectal neoplasms underwent L-TAMIS or R-TAMIS by two colorectal surgeons from January 2012 to April 2017. We retrospectively reviewed a prospectively maintained database to analyze demographics, peri-operative data, pathology, post-operative complications, and cost. RESULTS: There were no significant differences between L- and R-TAMIS on patient demographics. R-TAMIS showed a statically significant increase in cost of surgery by $880. Median direct cost of L-TAMIS was $3562 compared to $4440.92 for R-TAMIS (p = 0.04). Wider range of total duration for L-TAMIS is likely due to the variability of body habitus and location of rectal neoplasm, which can significantly limit L-TAMIS compare to R-TAMIS. There was a trend toward decreased blood loss in the R-TAMIS group. Mortality was 0% in both groups. CONCLUSIONS: After reviewing our experience, we conclude there is no significant difference between L- and R-TAMIS other than total direct cost. We confirmed that both L- and R-TAMIS are safe and associated with low morbidity. The limitations of this study include its small sample size. In the future, we hope to show promising data on R-TAMIS with increased sample size and experience, which may allow for transanal resection not previously feasible. Studies with long-term follow-up assessing oncological and functional results will be mandatory.

Differential Vulnerability of CA1 versus CA3 Pyramidal Neurons After Ischemia: Possible Relationship to Sources of Zn2+ Accumulation and Its Entry into and Prolonged Effects on Mitochondria.

Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia. However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca2+ entry, it is apparent that Zn2+ also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular Zn2+-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca2+ and Zn2+ accumulation in both CA1 and CA3, as indicated by the ability of Zn2+ chelators or Ca2+ entry blockers to delay pyramidal neuronal death in both regions. However, using knock-out animals (of MT-III and vesicular Zn2+ transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn2+ sources, with critical contributions of presynaptic release and its permeation through Ca2+- (and Zn2+)-permeable AMPA channels in CA3 and Zn2+ mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn2+ accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn2+ rises persisted for 10-30 min after OGD, followed by recovery over ∼40-60 min. Furthermore, the recovery appeared to be accompanied by mitochondrial Zn2+ accumulation (via the mitochondrial Ca2+ uniporter MCU) in CA1 but not in CA3 neurons and was markedly diminished in MT-III knock-outs, suggesting that it depended upon Zn2+ mobilization from this protein. SIGNIFICANCE STATEMENT: The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca2+ and Zn2+ contributing more in CA3, but with delayed and long-lasting accumulation of Zn2+ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn2+ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.

The effects of a heating pad on anxiety, pain, and distress during urodynamic study in the female patients with stress urinary incontinence.

AIMS: Although generally well tolerated, a urodynamic study is an unpleasant and stressful procedure for some patients. This study evaluated the effects of a heating pad on anxiety, pain, and distress during urodynamic studies in female patients with stress urinary incontinence. METHODS: A total of 74 female patients with stress urinary incontinence who underwent a urodynamic study between May 2015 and October 2015 were randomized to either the experimental group using a heating pad (n = 37) or control group (n = 37). In the experimental group, a heating pad was applied on the patient's sacrum during the urodynamic study. All patients completed the State-Trait Anxiety Inventory (20-80) before and after the procedure and assessed their degree of pain and distress after the procedure by the visual analog scale (0-10). Systolic and diastolic blood pressure and pulse rate were also checked before and after the procedure. RESULTS: Demographic characteristics, mean age, procedure duration, pre and post-procedural systolic, and diastolic blood pressures, and pulse rate were statistically similar between the experimental and control groups. The mean State-Trait Anxiety Inventory was significantly lower in the experimental group than in the control group (30.9 ± 7.5 vs 42.5 ± 10.1, P < 0.001). The experimental group showed significantly lower pain and distress scores (Visual Analog Scale, 2.7 ± 1.5, 3.0 ± 1.5) compared with the control group (4.0 ± 1.6, 4.7 ± 2.0, both P < 0.001). CONCLUSIONS: Using a heating pad for female patients with stress urinary incontinence during a urodynamic study is a simple, economical, and effective therapy that enhances patient comfort and decreases anxiety, pain, and distress.

Efficacy, safety and albuminuria-reducing effect of gemigliptin in Korean type 2 diabetes patients with moderate to severe renal impairment: A 12-week, double-blind randomized study (the GUARD Study).

AIMS: This multicentre, randomized, double-blind study investigated the efficacy and safety of gemigliptin in Korean type 2 diabetes mellitus (T2DM) patients with moderate to severe renal impairment (RI). METHODS: The study comprised a 12-week main part and a 40-week extension. We report here the results from the main part. In total, 132 patients were randomized to receive gemigliptin (n = 66) or placebo (n = 66). Changes in glycated haemoglobin (HbA1c; primary endpoint), other glycaemic control parameters (fasting plasma glucose, glycated albumin and fructosamine), lipid profiles, renal function parameters and safety profiles were evaluated. RESULTS: Baseline characteristics were comparable between the groups (mean HbA1c, 8.4% [68 mmol/mol]; age, 62.0 years; duration of type 2 diabetes, 16.3 years; estimated glomerular filtration rate, 33.3 mL/min/1.73 m2 ). At Week 12, the adjusted mean change ± standard error in HbA1c with gemigliptin was -0.82% ± 0.14% (-8.9 ± 1.5 mmol/mol), whereas it was 0.38% ± 0.14% (4.2 ± 1.5 mmol/mol) with placebo (significant between-group difference, P < .001). Other glycaemic control parameters showed beneficial changes as well. Body weight change (gemigliptin, -0.3 kg; placebo, -0.2 kg) was not significant. In the gemigliptin group, the mean decrease in urinary albumin creatinine ratio (UACR) was significant, both in patients with microalbuminuria (-41.9 mg/g creatinine, P = .03) and macroalbuminuria (-528.9 mg/g creatinine, P < .001). Drug-related adverse events were similar with gemigliptin and placebo (15% and 12%, respectively). CONCLUSIONS: A 12-week treatment with gemigliptin improved glycaemic control and provided UACR reduction in T2DM patients with moderate to severe RI. Gemigliptin was well tolerated, with no additional risk of hypoglycaemia and change in body weight.

Early Magnesium Treatment After Aneurysmal Subarachnoid Hemorrhage: Individual Patient Data Meta-Analysis.

BACKGROUND AND PURPOSE: Delayed cerebral ischemia (DCI) is an important cause of poor outcome after aneurysmal subarachnoid hemorrhage (SAH). Trials of magnesium treatment starting <4 days after symptom onset found no effect on poor outcome or DCI in SAH. Earlier installment of treatment might be more effective, but individual trials had not enough power for such a subanalysis. We performed an individual patient data meta-analysis to study whether magnesium is effective when given within different time frames within 24 hours after the SAH. METHODS: Patients were divided into categories according to the delay between symptom onset and start of the study medication: <6, 6 to 12, 12 to 24, and >24 hours. We calculated adjusted risk ratios with corresponding 95% confidence intervals for magnesium versus placebo treatment for poor outcome and DCI. RESULTS: We included 5 trials totaling 1981 patients; 83 patients started treatment<6 hours. For poor outcome, the adjusted risk ratios of magnesium treatment for start <6 hours were 1.44 (95% confidence interval, 0.83-2.51); for 6 to 12 hours 1.03 (0.65-1.63), for 12 to 24 hours 0.84 (0.65-1.09), and for >24 hours 1.06 (0.87-1.31), and for DCI, <6 hours 1.76 (0.68-4.58), for 6 to 12 hours 2.09 (0.99-4.39), for 12 to 24 hours 0.80 (0.56-1.16), and for >24 hours 1.08 (0.88-1.32). CONCLUSIONS: This meta-analysis suggests no beneficial effect of magnesium treatment on poor outcome or DCI when started early after SAH onset. Although the number of patients was small and a beneficial effect cannot be definitively excluded, we found no justification for a new trial with early magnesium treatment after SAH.

New teleomorph combinations in the entomopathogenic genus Metacordyceps.

The genus Metacordyceps contains arthropod pathogens in Clavicipitaceae (Hypocreales) that formerly were classified in Cordyceps sensu Kobayasi et Mains. Of the current arthropod pathogenic genera of Hypocreales, the genus Metacordyceps remains one of the most poorly understood and contains a number of teleomorphic morphologies convergent with species of Cordyceps s.s. (Cordycipitaceae) and Ophiocordyceps (Ophiocordycipitaceae). Of note, the anamorph genera Metarhizium and Pochonia were found to be associated only with Metacordyceps and demonstrated to be phylogenetically informative for the clade. Several species of Cordyceps considered to have uncertain placements (incertae sedis) in the current taxonomic framework of clavicipitoid fungi were collected during field expeditions mostly in eastern Asia. Species reclassified here in Metacordyceps include Cordyceps atrovirens Kobayasi & Shimizu, Cordyceps indigotica Kobayasi & Shimizu, Cordyceps khaoyaiensis Hywel-Jones, Cordyceps kusanagiensis Kobayasi & Shimizu, Cordyceps martialis Speg., Ophiocordyceps owariensis Kobayasi, Cordyceps pseudoatrovirens Kobayasi & Shimizu and Ophicordyceps owariensis f. viridescens (Uchiy. & Udagawa) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora. Incorporation of these species in a multigene phylogenetic framework of the major clades of clavicipitoid fungi more than doubled the number of species in Metacordyceps and allowed for refinement of morphological concepts for the genus consistent with the phylogenetic structure. Based on these findings we then discuss evolution of this genus, subgeneric relationships, anamorph connections, and suggest additional species that should be confirmed for possible inclusion in Metacordyceps.

Facile discovery of a therapeutic agent for NK-mediated synergistic antitumor effects using a patient-derived 3D platform.

Despite the essential roles of natural killer (NK) cells in cancer treatment, the physical barrier and biological cues of the tumor microenvironment (TME) may induce NK cell dysfunction, causing their poor infiltration into tumors. The currently available two-dimensional (2D) cancer-NK co-culture systems hardly represent the characteristics of TME and are not suitable for tracking the infiltration of immune cells and assessing the efficacy of immunotherapy. This study aims to monitor NK-mediated cancer cell killing using a polymer thin film-based, 3D assay platform that contains highly tumorigenic cancer spheroids. A poly(cyclohexyl methacrylate) (pCHMA)-coated surface enables the generation of tumorigenic spheroids from pancreatic cancer patient-derived cancer cells, showing considerable amounts of extracellular matrix (ECM) proteins and cancer stem cell (CSC)-like characteristics. The 3D spheroid-based assay platform allows rapid discovery of a therapeutic agent for synergistic NK-mediated cytotoxicity through imaging-based high-content screening. In detail, the small molecule C19, known as a multi-epithelial-mesenchymal transition pathway inhibitor, is shown to enhance NK activation and infiltration via modulation of the ECM, resulting in synergistic cytotoxicity against cancer spheroids. This 3D biomimetic co-culture assay platform provides promising applications for predicting patient-specific responses to immunotherapy through advanced therapeutic combinations involving a chemical drug and immune cells.

Biosynthesis of a new UDP-sugar, UDP-2-acetamido-2-deoxyxylose, in the human pathogen Bacillus cereus subspecies cytotoxis NVH 391-98.

We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDP-XylNAc from UDP-GlcNAcA in the presence of exogenous NAD(+). UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mM(-1) s(-1). The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease.

Biosynthesis of UDP-xylose and UDP-arabinose in Sinorhizobium meliloti 1021: first characterization of a bacterial UDP-xylose synthase, and UDP-xylose 4-epimerase.

Sinorhizobium meliloti is a soil bacterium that fixes nitrogen after being established inside nodules that can form on the roots of several legumes, including Medicago truncatula. A mutation in an S. meliloti gene (lpsB) required for lipopolysaccharide synthesis has been reported to result in defective nodulation and an increase in the synthesis of a xylose-containing glycan. Glycans containing xylose as well as arabinose are also formed by other rhizobial species, but little is known about their structures and the biosynthetic pathways leading to their formation. To gain insight into the biosynthesis of these glycans and their biological roles, we report the identification of an operon in S. meliloti 1021 that contains two genes encoding activities not previously described in bacteria. One gene encodes a UDP-xylose synthase (Uxs) that converts UDP-glucuronic acid to UDP-xylose, and the second encodes a UDP-xylose 4-epimerase (Uxe) that interconverts UDP-xylose and UDP-arabinose. Similar genes were also identified in other rhizobial species, including Rhizobium leguminosarum, suggesting that they have important roles in the life cycle of this agronomically important class of bacteria. Functional studies established that recombinant SmUxs1 is likely to be active as a dimer and is inhibited by NADH and UDP-arabinose. SmUxe is inhibited by UDP-galactose, even though this nucleotide sugar is not a substrate for the 4-epimerase. Unambiguous evidence for the conversions of UDP-glucuronic acid to UDP-α-D-xylose and then to UDP-ß-L-arabinose (UDP-arabinopyranose) was obtained using real-time (1)H-NMR spectroscopy. Our results provide new information about the ability of rhizobia to form UDP-xylose and UDP-arabinose, which are then used for the synthesis of xylose- and arabinose-containing glycans.

Design and fabrication of label-free biochip using a guided mode resonance filter with nano grating structures by injection molding process.

This paper introduces technology to fabricate a guided mode resonance filter biochip using injection molding. Of the various nanofabrication processes that exist, injection molding is the most suitable for the mass production of polymer nanostructures. Fabrication of a nanograting pattern for guided mode resonance filters by injection molding requires a durable metal stamp, because of the high injection temperature and pressure. Careful consideration of the optimized process parameters is also required to achieve uniform sub-wavelength gratings with high fidelity. In this study, a metallic nanostructure pattern to be used as the stamp for the injection molding process was fabricated using electron beam lithography, a UV nanoimprinting process, and an electroforming process. A one-dimensional nanograting substrate was replicated by injection molding, during which the process parameters were controlled. To evaluate the geometric quality of the injection molded nanograting patterns, the surface profile of the fabricated nanograting for different processing conditions was analyzed using an atomic force microscope and a scanning electron microscope. Finally, to demonstrate the feasibility of the proposed process for fabricating guided mode resonance filter biochips, a high-refractive-index material was deposited on the polymer nanograting and its guided mode resonance characteristics were analyzed.

In vivo confocal microscopy of equine fungal keratitis.

OBJECTIVE: To describe in vivo corneal confocal microscopy of horses with fungal keratitis and correlate findings with clinical, histopathological, and microbiological evaluations of clinical cases and an ex vivo experimental equine fungal keratitis model. ANIMALS STUDIED: A total of 12 horses with naturally-acquired fungal keratitis and ex vivo equine corneas experimentally infected with clinical fungal isolates. PROCEDURES: Horses with naturally-acquired fungal keratitis were examined with a modified Heidelberg Retina Tomograph II and Rostock Cornea Module. Confocal microscopy images of clinical isolates of Aspergillus fumigatus, Fusarium solani, and Candida albicans were obtained by examination of in vitro cultures and experimentally infected ex vivo equine corneas. RESULTS: Non-specific in vivo corneal confocal microscopic findings in horses with fungal keratitis included leukocyte infiltrates, activated keratocytes, anterior stromal dendritic cell infiltrates, and vascularization. Linear, branching, hyper-reflective structures that were 2-6 µm in width and 200 to >400 µm in length were detected in all horses with filamentous fungal keratitis. Round to oval hyper-reflective structures that were 2-8 µm in diameter were detected in a horse with yeast fungal keratitis. The in vivo confocal microscopic appearance of the organisms was consistent with fungal morphologies observed during examination of in vitro cultures and infected ex vivo equine corneas. CONCLUSIONS: In vivo corneal confocal microscopy is a rapid and non-invasive method of diagnosing fungal keratitis in the horse. This imaging technique is useful for both ulcerative and non-ulcerative fungal keratitis, and is particularly advantageous for confirming the presence of fungi in deep corneal stromal lesions.

The effect of topical ocular corticosteroid administration in dogs with experimentally induced latent canine herpesvirus-1 infection.

Recurrent herpes simplex virus-1 (HSV-1) ocular infection is a frequent cause of morbidity and blindness. Factors that trigger viral reactivation are poorly understood and the role of topical ocular corticosteroid administration in the development of recurrent HSV-1 ocular disease is not clear. Clinical reports and epidemiological studies suggested topical corticosteroids may reactivate latent HSV-1 and result in recrudescent ocular disease; however, experimental studies to establish this causal relationship produced inconsistent results. The previous experimental studies were performed by infecting unnatural host species with HSV-1 and aspects of viral behavior and reactivation within these animals may differ from the host for which the virus is adapted. The purpose of the study reported here was to determine if topical ocular corticosteroid administration results in viral reactivation and recrudescent ocular disease in a host-adapted pathogen animal model of HSV-1 recurrent ocular disease. Canine herpesvirus-1 (CHV-1) is a corticosteroid-sensitive alphaherpesvirus that is biologically related to HSV-1 and induces similar ocular lesions in canids during recurrent infection. A randomized, masked, placebo-controlled, crossover study was performed. Primary ocular CHV-1 infection was experimentally induced in mature specific pathogen-free beagles by topical ocular inoculation and the presence of reactivatable latency was later confirmed by administration of an immunosuppressive dosage of systemic corticosteroid to the dogs. Twelve months following experimental CHV-1 reactivation, dogs were administered either topical ocular prednisolone acetate (1.0% ophthalmic suspension, one drop in both eyes, four times daily) or placebo (artificial tear solution, one drop in both eyes, four times daily) for 28 days. After a 14 day washout period, the treatment groups were reversed and study agents administered for an additional 28 days. Ophthalmic examinations, in vivo ocular confocal microscopy, real-time quantitative CHV-1 polymerase chain reaction assays, and CHV-1 serum neutralization antibody titers were performed at regular intervals throughout the study. Viral reactivation was not detected in dogs administered topical ocular prednisolone or placebo as determined by clinical ocular disease recrudescence, in vivo ocular confocal microscopic findings, ocular viral shedding, and serologic response. Similar to other animal models of recurrent HSV-1 ocular infection, the behavior of latent CHV-1 in dogs may differ from HSV-1 in humans; however, results of the present study suggest administration of topical ocular prednisolone at the evaluated drug concentration, dosing frequency, and treatment duration is not likely to result in detectable reactivation of latent CHV-1 in experimentally infected dogs. This may be attributed to insufficient systemic absorption of locally administered corticosteroid to reactivate latent virus and produce recurrent disease. Crystalline corneal opacities that were apparently not associated with viral reactivation were detected by clinical examination and in vivo confocal microscopy in two dogs during topical ocular prednisolone administration. The crystalline keratopathy may have resulted from corneal degeneration associated with metaherpetic disease, corticosteroid administration, or a combination of both factors.

Zn2+ entry through the mitochondrial calcium uniporter is a critical contributor to mitochondrial dysfunction and neurodegeneration.

Excitotoxic Ca2+ accumulation contributes to ischemic neurodegeneration, and Ca2+ can enter the mitochondria through the mitochondrial calcium uniporter (MCU) to promote mitochondrial dysfunction. Yet, Ca2+-targeted therapies have met limited success. A growing body of evidence has highlighted the underappreciated importance of Zn2+, which also accumulates in neurons after ischemia and can induce mitochondrial dysfunction and cell death. While studies have indicated that Zn2+ can also enter the mitochondria through the MCU, the specificity of the pore's role in Zn2+-triggered injury is still debated. Present studies use recently available MCU knockout mice to examine how the deletion of this channel impacts deleterious effects of cytosolic Zn2+ loading. In cultured cortical neurons from MCU knockout mice, we find significantly reduced mitochondrial Zn2+ accumulation. Correspondingly, these neurons were protected from both acute and delayed Zn2+-triggered mitochondrial dysfunction, including mitochondrial reactive oxygen species generation, depolarization, swelling and inhibition of respiration. Furthermore, when toxic extramitochondrial effects of Ca2+ entry were moderated, both cultured neurons (exposed to Zn2+) and CA1 neurons of hippocampal slices (subjected to prolonged oxygen glucose deprivation to model ischemia) from MCU knockout mice displayed decreased neurodegeneration. Finally, to examine the therapeutic applicability of these findings, we added an MCU blocker after toxic Zn2+ exposure in wildtype neurons (to induce post-insult MCU blockade). This significantly attenuated the delayed evolution of both mitochondrial dysfunction and neurotoxicity. These data-combining both genetic and pharmacologic tools-support the hypothesis that Zn2+ entry through the MCU is a critical contributor to ischemic neurodegeneration that could be targeted for neuroprotection.

Acanthamoeba sclerokeratitis in a cat.

CASE DESCRIPTION: A 12-year-old neutered male domestic shorthair cat with chronic anterior uveitis and secondary glaucoma of the right eye was examined for persistent blepharospasm 2 weeks after corneal debridement and grid keratotomy for nonhealing superficial ulcerative keratitis. CLINICAL FINDINGS: Examination of the right eye revealed a central superficial corneal ulcer associated with corneal epithelial and subepithelial infiltrates and mild aqueous flare. Structures consistent with amoeboid cysts and trophozoites were detected in the cornea by in vivo confocal microscopy. Suppurative keratitis was identified cytologically. An Acanthamoeba spp was isolated through culture and identified by a PCR assay of corneal specimens. TREATMENT AND OUTCOME: Symptomatic and antiamoebic (polyhexamethylene biguanide 0.02% ophthalmic solution) treatments were instituted. Over the following 6 weeks, the cat lost vision in the affected eye and lesions progressed to nonulcerative stromal keratitis associated with a dense paracentral corneal stroma ring infiltrate and anterior lens luxation. The globe was enucleated, and lymphoplasmacytic sclerokeratitis, anterior uveitis, and retinal detachment were noted. Acanthamoeba organisms were detected within the corneal stroma and anterior sclera with histologic and immunohistochemical stains. The amoebae were classified to the Acanthamoeba T4 genotype by DNA sequencing. The cat had no medical problems attributed to Acanthamoeba infection over 36 months after enucleation, until the cat was lost to follow-up. CLINICAL RELEVANCE: Naturally acquired Acanthamoeba sclerokeratitis is described in a cat for the first time. Acanthamoeba infection should be considered for cats with superficial corneal disease refractory to appropriate treatments and especially occurring after ocular trauma, including keratotomy.

Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation.

We examined the effect of ice-binding protein derived from Leucosporidium (LeIBP) on the cryopreservation of bull semen and compared it with that derived from previously reported Antifreeze Protein III (AFPIII). Six concentrations of LeIBP (10-1  ~ 104  µg/ml) and AFPIII (10-1  ~ 104  µg/ml) were added to the bull semen extender, respectively. Sperm kinematic parameters were measured to examine sperm toxicity and cryopreserved sperm quality. Measures of antioxidant activity such as superoxide dismutase (SOD), reduced glutathione/oxidative glutathione (GSH/GSSG), and total antioxidant capacity (TAC) were analysed to identify the effect of LeIBP on sperm quality. In addition, sperm viability was analysed using a flow cytometer and fluorescence microscope by SYBR14/PI staining. The results showed that the LeIBP groups (0.1, 1 and 10 µg/ml) were less toxic, and the quality of the sperm were dramatically improved in the extenders containing 0.1 µg/ml LeIBP among concentrations of LeIBP and AFPIII. The SOD activity of LeIBP was greater than that of AFPIII and control. In addition, sperm viability was enhanced in the LeIBP-treated group. In summary, LeIBP is a useful cryoprotective adjuvant for bull sperm cryopreservation, and the most efficient concentration of LeIBP is 0.1 µg/ml.

Genotyping and phylogenetic analysis of bovine viral diarrhea virus isolates from BVDV infected alpacas in North America.

Over a three-year period, 2004-2007, greater than 12,000 alpacas in the United States were screened by real-time RT-PCR to identify alpacas persistently infected (PI) with bovine viral diarrhea virus (BVDV). A total of 46 BVD viruses were isolated from PI alpacas or diagnostic samples from alpacas. Forty-three US alpaca BVDV isolates and 3 Canadian isolates were analyzed by comparison of nucleotide sequences of two viral genomic regions, the 5'-UTR and the N(pro) gene to determine their genetic relatedness. All 46 alpaca BVDV isolates from 8 different states of the US and Canada were genotype 1b with > or =99% nt identity in the 290-base 5'-UTR region with the exception of one Canadian isolate. In contrast, 21 bovine BVDV isolates collected during the same period were grouped into the typical 3 genotypes, 1a, 1b, and 2, respectively. Forty five alpaca BVDV isolates formed a distinctive cluster separated from closely related bovine genotype 1b isolates by phylogenetic analysis of the 5'-UTR region. Comparison of the 504-base N(pro) gene sequences of 32 alpaca isolates also assigned them all to type 1b in a similar fashion as observed with the 5'-UTR region. The results suggest that unique genotypes of bovine BVDV 1b may be maintained in the alpaca population even though camelids are susceptible to infection by other genotypes. Further studies are needed to address why alpacas were predominantly infected with genotype 1b BVDV isolates and how bovine BVD viruses evolved to infect alpacas.

Outbreak of ocular disease associated with naturally-acquired canine herpesvirus-1 infection in a closed domestic dog colony.

OBJECTIVE: To describe clinical and virological findings of an outbreak of ocular disease attributed to naturally-acquired primary canine herpesvirus-1 (CHV-1) infection in a closed domestic dog colony. ANIMALS STUDIED: Twenty-seven 10- to 16-week-old laboratory Beagles. PROCEDURE: Complete ophthalmic examinations were performed and ocular samples collected for CHV-1 polymerase chain reaction and virus isolation. RESULTS: The prevalence of ocular morbidity was 100% in examined dogs. Lesions were restricted to the ocular surface and included bilateral conjunctivitis (100% of dogs); punctate, dendritic, or geographic ulcerative keratitis (26% of dogs); and non-ulcerative keratitis (19% of dogs). Conjunctival petechiae were detected in 22% of dogs. Punctate and dendritic corneal ulcers were frequently organized into discrete groups or linear arrangements. Non-ulcerative keratitis appeared clinically as a perilimbal ring of superficial corneal vascularization and leukocyte infiltration. CHV-1 was detected in ocular samples by polymerase chain reaction or virus isolation in all dogs sampled. CONCLUSIONS: In susceptible populations of domestic dogs, CHV-1 may be associated with outbreaks of highly contagious ocular infection in the absence of concurrent overt systemic disease. This naturally-acquired outbreak of CHV-1 infection provides an opportunity to report the spectrum and prevalence of ocular lesions associated with primary ocular CHV-1 infection in dogs. Conjunctivitis was the most frequent ocular lesion detected. Ulcerative and non-ulcerative keratitis were less prevalent and of variable clinical appearance. Dendritic ulcerative keratitis, a classic and relatively specific ocular lesion associated with alphaherpesvirus infection, was detected in < 20% of dogs.

Experimental primary ocular canine herpesvirus-1 infection in adult dogs.

OBJECTIVE-To characterize clinical ocular disease, viral shedding, and serologic response associated with primary canine herpesvirus-1 (CHV-1) ocular infection in naïve adult dogs. ANIMALS-12 specific pathogen-free adult Beagles. PROCEDURES-Dogs were topically inoculated in the right eye with CHV-1 (infection group; n = 8) or virus-free medium (control group; 4). Dogs were inoculated with or without corneal microtrephination and subconjunctivally administered corticosteroids. Conjunctiva, buffy coat, and serum samples for real-time PCR assay, virus isolation, and serum neutralization (SN) antibody titers were collected until postinfection day (PID) 224, and general physical and ophthalmologic examinations were performed. RESULTS-Dogs in the infection group developed bilateral, mild to moderate conjunctivitis that reached maximal intensity on PIDs 7 to 10. Ocular viral shedding was detected in all dogs in the infection group between PIDs 3 and 10. Infected dogs developed CHV-1 SN antibody titers, beginning at PID 7 and peaking on PID 21. All buffy coat PCR assay results were negative. Corneal microtrephination and subconjunctival corticosteroid administration did not significantly affect clinical disease or viral shedding. Following recovery from primary infection, dogs remained clinically normal, did not shed virus, and had slowly decreasing SN antibody titers. Dogs in the control group did not develop conjunctivitis, shed virus, or develop CHV-1 SN antibody titers. CONCLUSIONS AND CLINICAL RELEVANCE-Primary ocular infection of adult dogs with CHV-1 was associated with self-limiting conjunctivitis and ocular viral shedding, which was evident in the absence of clinically detectable keratitis or systemic disease. Features of this infection resembled herpes simplex virus primary ocular infection in humans.

Mitochondrial Zn2+ Accumulation: A Potential Trigger of Hippocampal Ischemic Injury.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca2+ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn2+, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn2+ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn2+ effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn2+ contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn2+ and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn2+-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.