The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Silver Nanoparticles Complexed with Bovine Submaxillary Mucin Possess Strong Antibacterial Activity and Protect against Seedling Infection.

A simple method for the synthesis of nanoparticles (NPs) of silver (Ag) in a matrix of bovine submaxillary mucin (BSM) was reported previously by some of the authors of this study. Based on mucin characteristics such as long-lasting stability, water solubility, and surfactant and adhesive characteristics, we hypothesized that these compounds, named BSM-Ag NPs, may possess favorable properties as potent antimicrobial agents. The goal of this study was to assess whether BSM-Ag NPs possess antibacterial activity, focusing on important plant-pathogenic bacterial strains representing both Gram-negative (Acidovorax and Xanthomonas) and Gram-positive (Clavibacter) genera. Growth inhibition and bactericidal assays, as well as electron microscopic observations, demonstrate that BSM-Ag NPs, at relatively low concentrations of silver, exert strong antimicrobial effects. Moreover, we show that treatment of melon seeds with BSM-Ag NPs effectively prevents seed-to-seedling transmission of Acidovorax citrulli, one of the most threatening pathogens of cucurbit production worldwide. Overall, our findings demonstrate strong antimicrobial activity of BSM-Ag NPs and their potential application for reducing the spread and establishment of devastating bacterial plant diseases in agriculture.IMPORTANCE Bacterial plant diseases challenge agricultural production, and the means available to manage them are limited. Importantly, many plant-pathogenic bacteria have the ability to colonize seeds, and seed-to-seedling transmission is a critical route by which bacterial plant diseases spread to new regions and countries. The significance of our study resides in the following aspects: (i) the simplicity of the method of BSM-Ag NP synthesis, (ii) the advantageous chemical properties of BSM-Ag NPs, (iii) the strong antibacterial activity of BSM-Ag NPs at relatively low concentrations of silver, and (iv) the fact that, in contrast to most studies on the effects of metal NPs on plant pathogens, the proof of concept for the novel compound is supported by in planta assays. Application of this technology is not limited to agriculture; BSM-Ag NPs potentially could be exploited as a potent antimicrobial agent in a wide range of industrial areas, including medicine, veterinary medicine, cosmetics, textiles, and household products.

Minocycline upregulates pro-survival genes and downregulates pro-apoptotic genes in experimental glaucoma.

PURPOSE: Minocycline, a second-generation tetracycline with anti-inflammatory and anti-apoptotic properties, was reported to be neuroprotective in experimental glaucoma and optic nerve transection as well as in other neurodegenerative diseases. The purpose of this study was to investigate the mechanism underlying that neuroprotective effect in murine glaucoma. METHODS: Elevated intraocular pressure was induced in 159 rats by the translimbal photocoagulation laser model. Minocycline 22 mg/kg or saline was injected intraperitoneally starting 3 days before the induction of glaucoma, and continued daily until the animals were sacrificed. The effect of minocycline on gene expression was evaluated using a quantitative polymerase chain reaction (PCR) array for apoptosis. The involvement of selected pro-apoptotic, pro-survival, and inflammatory genes was further analyzed by quantitative real-time PCR at multiple time points. Immunohistochemistry was used to study the effect of minocycline on microglial activation and to localize Bcl-2 changes. RESULTS: Minocycline significantly increased the anti-apoptotic gene Bcl-2 expression at day 8 and day 14 after the induction of glaucoma (p = 0.04 and p = 0.03 respectively), and decreased IL-18 expression in the retina at day 14 and day 30 (p = 0.04 and p < 0.001 respectively). PCR arrays suggested that additional genes were affected by minocycline, including Tp53bp2, TRAF4, osteoprotegerin, caspase 1 and 4, and members of the tumor necrosis factor superfamily. Additionally, minocycline decreased the amount of activated microglia in glaucomatous eyes. CONCLUSIONS: These results suggest that minocycline upregulates pro-survival genes and downregulates apoptotic genes, thus shifting the balance toward the anti-apoptotic side in experimental glaucoma.

Minocycline mechanism of neuroprotection involves the Bcl-2 gene family in optic nerve transection.

The second-generation tetracycline, minocycline, has been shown to exhibit neuroprotective therapeutic benefits in many neurodegenerative diseases including experimental glaucoma and optic nerve transection (ONT). This study investigated the mechanism underlying minocycline neuroprotection in a model of ONT. ONT was applied unilaterally in 36 Wistar rat eyes. The rats were randomly divided into a minocycline (22 mg/kg/d) treatment group and a saline treatment group (control). Treatment (minocycline or saline) was given by intraperitoneal injections initiated 3 d before ONT and continued daily until the end of the experiment. The involvement of pro-apoptotic, pro-survival and inflammatory pathways was analyzed by quantitative Real-Time Polymerase Chain Reaction at 4 h and 3 d after the transection in both treatment groups. The involvement of Bcl-2 protein was evaluated by immunohistochemistry. We found that Minocycline significantly increased the expression of the antiapoptotic gene bcl-2 4 h after transection (n = 8, p = 0.008) and decreased the expression of Bax at the same time point (n = 8, p = 0.03). Tumor Necrosis Factor α (TNFα), Inhibitor of Apoptosis Protein (IAP1) and Gadd45α were significantly upregulated in the retinas of eyes with ONTs compared to control (n = 10 for each gene, p = 0.02, p = 0.03, p = 0.04, respectively) but this effect was unaffected by minocycline. This study further support that the mechanism underlying minocycline neuroprotection involves the Bcl-2 gene family, suggesting that minocycline has antiapoptotic properties that support its value as a promising neuroprotective drug.

Comparison between axonal and retinal ganglion cell gene expression in various optic nerve injuries including glaucoma.

PURPOSE: The pathogenesis of retinal ganglion cell loss in glaucoma remains incompletely understood. Current evidence suggests that the optic nerve (ON) head and axons are the main site of injury in glaucoma. This study compares changes in prosurvival and proapoptotic gene expression in ONs with those in retinas in three models of ocular injury, specifically ON transection (ONTX), N-methyl-D-aspartate (NMDA) retinal toxicity, and experimental glaucoma. METHODS: Rats (n=240) were divided into three models (ONTX, NMDA retinal toxicity, and experimental glaucoma). The experimental model was induced unilaterally and the contralateral eye served as control. Rats were sacrificed at 4-5 different time points specific for each model. ONs and retinas were isolated for real-time PCR investigation of expression of selected genes. Immunohistochemistry localized changes in inhibitor of apoptosis (IAP)-1 and X-linked IAP (XIAP) proteins in retinas and ONs. Colocalization was measured using Imaris colocalization software (three-dimensional analysis). RESULTS: The earliest changes in gene expression occurred in ONs in the ONTX model and in retinas in the NMDA model, as expected. However, some gene changes occurred first in ONs, while others occurred first in retinas in the glaucoma model. The expression patterns of the prosurvival genes IAP-1 and XIAP differed between retinas and ONs of glaucomatous eyes: Both were upregulated in the retinas, but XIAP was downregulated in the ONs, while IAP-1 stayed unchanged. Colocalization of IAP-1, XIAP, glial fibrillary acidic protein, and Thymus cell antigen-1 (Thy-1) suggested that IAP-1 was colocalized mostly with Thy-1 and XIAP with glial fibrillary acidic protein in the ONs. Members of the B-cell lymphoma 2 (BCL-2) family were similarly involved in the ONs and retinas of glaucomatous, transected, and NMDA-injected eyes. The expression of the prosurvival genes, Bcl-2 and Bcl-xl, decreased significantly in both the ONs and retinas of injured eyes. The proapoptotic genes, BCL2-associated X protein (BAX) and Bcl-2-associated death promoter (BAD), were significantly upregulated in both injured retinas and ONs. CONCLUSIONS: The overexpression of XIAP and IAP-1 genes in the retinas was not associated with similar changes in the ONs of glaucomatous eyes. The lack of activation of these prosurvival genes in the ONs may explain the increased vulnerability of ONs to elevated intraocular pressure.

Increase in retinal ganglion cells' susceptibility to elevated intraocular pressure and impairment of their endogenous neuroprotective mechanism by age.

PURPOSE: To investigate age-associated changes in retinal ganglion cell (RGC) response to elevated intraocular pressure (IOP), and to explore the mechanism underlying these changes. Specifically, the effect of aging on inhibitor of apoptosis (IAP) gene family expression was investigated in glaucomatous eyes. METHODS: IOP was induced unilaterally in 82 Wistar rats using the translimbal photocoagulation laser model. IOP was measured using a TonoLab tonometer. RGC survival was evaluated in 3-, 6-, 13-, and 18-month-old animals. Changes in the RNA profiles of young (3-month-old) and old glaucomatous retinas were examined by PCR array for apoptosis; changes in selected genes were validated by real-time PCR; and changes in selected proteins were localized by immunohistochemistry. RESULTS: There were no significant IOP differences between the age groups. However, there was a natural significant loss of RGCs with aging and this was more prevalent in glaucomatous eyes. The number of RGCs in glaucomatous eyes decreased from 669±123 RGC/mm² at 3 months to 486±114 RGC/mm² at 6 months and 189±46.5 RGC/mm² at 18 months (n=4-8, p=0.048, analysis of variance). The PCR array revealed different changes in proapoptotic and prosurvival genes between young and old eyes. The two important prosurvival genes, IAP-1 and X-linked IAP (XIAP), acted in opposite directions in 3-month-old and 15-month-old rats, and were significantly decreased in aged glaucomatous retinas, while their expression increased significantly in young glaucomatous eyes. P53 levels did not vary between young glaucomatous and normal fellow eyes, but were reduced with age. B-cell leukemia/lymphoma 2 (Bcl-2) family members and tumor necrosis factor (TNF)-α expression were unaffected by age. Immunohistochemistry results suggested that the sources of changes in IAP-1 protein expression are RGCs and glial cells, and that most XIAP secretion comes from RGCs. CONCLUSIONS: Decreased IAP-1 and XIAP gene expression in aged eyes may predispose RGCs to increased vulnerability to glaucomatous damage. These findings suggest that aging impairs the endogenous neuroprotective mechanism of RGCs evoked by elevated IOP.

Phenotypic variation in the plant pathogenic bacterium Acidovorax citrulli.

Acidovorax citrulli causes bacterial fruit blotch (BFB) of cucurbits, a disease that threatens the cucurbit industry worldwide. Despite the economic importance of BFB, little is known about pathogenicity and fitness strategies of the bacterium. We have observed the phenomenon of phenotypic variation in A. citrulli. Here we report the characterization of phenotypic variants (PVs) of two strains, M6 and 7a1, isolated from melon and watermelon, respectively. Phenotypic variation was observed following growth in rich medium, as well as upon isolation of bacteria from inoculated plants or exposure to several stresses, including heat, salt and acidic conditions. When grown on nutrient agar, all PV colonies possessed a translucent appearance, in contrast to parental strain colonies that were opaque. After 72 h, PV colonies were bigger than parental colonies, and had a fuzzy appearance relative to parental strain colonies that are relatively smooth. A. citrulli colonies are generally surrounded by haloes detectable by the naked eye. These haloes are formed by type IV pilus (T4P)-mediated twitching motility that occurs at the edge of the colony. No twitching haloes could be detected around colonies of both M6 and 7a1 PVs, and microscopy observations confirmed that indeed the PVs did not perform twitching motility. In agreement with these results, transmission electron microscopy revealed that M6 and 7a1 PVs do not produce T4P under tested conditions. PVs also differed from their parental strain in swimming motility and biofilm formation, and interestingly, all assessed variants were less virulent than their corresponding parental strains in seed transmission assays. Slight alterations could be detected in some DNA fingerprinting profiles of 7a1 variants relative to the parental strain, while no differences at all could be seen among M6 variants and parental strain, suggesting that, at least in the latter, phenotypic variation is mediated by slight genetic and/or epigenetic alterations.