Reduced endothelial caveolin-1 underlies deficits in brain insulin signalling in type 2 diabetes.

Patients with type 2 diabetes exhibit severe impairments in insulin signalling in the brain and are five times more likely to develop Alzheimer's disease. However, what leads to these impairments is not fully understood. Here, we show reduced expression of endothelial cell caveolin-1 (Cav-1) in the db/db (Leprdb) mouse model of type 2 diabetes. This reduction correlated with alterations in insulin receptor expression and signalling in brain microvessels as well as brain parenchyma. These findings were recapitulated in the brains of endothelial cell-specific Cav-1 knock-out (Tie2Cre; Cav-1fl/fl) mice. Lack of Cav-1 in endothelial cells led to reduced response to insulin as well as reduced insulin uptake. Furthermore, we observed that Cav-1 was necessary for the stabilization of insulin receptors in lipid rafts. Interactome analysis revealed that insulin receptor interacts with Cav-1 and caveolae-associated proteins, insulin-degrading enzyme and the tight junction protein Zonula Occludence-1 in brain endothelial cells. Restoration of Cav-1 in Cav-1 knock-out brain endothelial cells rescued insulin receptor expression and localization. Overall, these results suggest that Cav-1 regulates insulin signalling and uptake by brain endothelial cells by modulating IR-α and IR-ß localization and function in lipid rafts. Furthermore, depletion of endothelial cell-specific Cav-1 and the resulting impairment in insulin transport leads to alteration in insulin signalling in the brain parenchyma of type 2 diabetics.

In Vitro and In Vivo Activity, Tolerability, and Mechanism of Action of BX795 as an Antiviral against Herpes Simplex Virus 2 Genital Infection.

Herpes simplex virus type 2 (HSV-2) causes recurrent lesions in the anogenital area that may be transmitted through sexual encounters. Nucleoside analogs, such as acyclovir (ACV), are currently prescribed clinically to curb this infection. However, in some cases, reduced efficacy has been observed due to the emergence of resistance against these drugs. In our previous study, we reported the discovery of a novel anti-HSV-1 small molecule, BX795, which was originally used as an inhibitor of TANK-binding kinase 1 (TBK1). In this study, we report the antiviral efficacy of BX795 on HSV-2 infection in vaginal epithelial cells in vitro at 10 µM and in vivo at 50 µM. Additionally, through biochemical assays in vitro and histopathology in vivo, we show the tolerability of BX795 in vaginal epithelial cells at concentrations as high as 80 µM. Our investigations also revealed that the mechanism of action of BX795 antiviral activity stems from the reduction of viral protein translation via inhibition of protein kinase B phosphorylation. Finally, using a murine model of vaginal infection, we show that topical therapy using 50 µM BX795 is well tolerated and efficacious in controlling HSV-2 replication.

OPTN-TBK1 axis and a role for PLK1 in HSV-1 infection.

IMPORTANCE: Herpes simplex virus type 1 (HSV-1) is globally prevalent, with latent infections observed in up to 80% of the population. The virus is known for subverting host defense mechanisms and infiltrating the nervous system to establish latency in peripheral ganglia. Multiple stressors can reactivate the virus, and recurrent herpes has been linked to vision loss and neurodegeneration. Identifying critical host factors that limit the spread of HSV-1 and the subsequent establishment of latent infection holds the potential to drive new intervention strategies for eradicating the virus. Numerous pieces of evidence underscore the significance of Tank-binding kinase 1 (TBK1) in restricting HSV-1. Reports have also suggested that phosphorylation of optineurin (OPTN) by TBK1 is required for triggering OPTN-mediated autophagy for HSV degradation. This report adds new insights into the roles of OPTN and TBK1 in HSV-1 infection and provides proof of a TBK1-independent HSV-1 restriction through OPTN. It confirms that TBK1 activation can be substituted by PLK1 to provide protection against HSV-1. In contrast, the activation of OPTN is likely an indispensable host defense mechanism for optimal defense against HSV-1.

Unique Attributes of Guinea Pigs as New Models to Study Ocular Herpes Pathophysiology and Recurrence.

Purpose: The objective of this study was to explore the ocular and systemic outcomes of herpes simplex virus type 1 (HSV-1) infection in guinea pigs, to monitor the spontaneous reactivation of the virus, and to assess the effectiveness of various treatments, drawing comparisons to conventional rabbit models. Methods: Guinea pigs and rabbits were infected in the right corneas with differing doses and strains of HSV-1. Observations were made over a 71-day period, focusing on comparing ocular lesions, viral shedding patterns, and weight loss between the two animal models. Postinfection, the effectiveness of trifluridine ophthalmic drops, oral acyclovir, and valacyclovir was evaluated. The confirmation of viral infection was done through virus titer assay, fluorescein staining, and corneal imaging. Results: Guinea pigs and rabbits manifested symptoms akin to human herpes stromal keratitis (HSK) when exposed to varying titers of viral suspension. Regardless of the initial viral load, all guinea pig groups demonstrated comparable ocular pathology, witnessing conditions like blepharitis and conjunctivitis within 3 days, progressing to severe conditions, including total corneal opacification and necrotizing keratitis. Tear film collection revealed nonsignificant differences in viral plaques between all groups. Notably, guinea pigs in the low-infection group experienced the most weight loss, although without significant differences. The replication of the same experiment on rabbits yielded consistent results in disease pathology across different groups, with occurrences of blepharitis and conjunctivitis. Interestingly, after initial resolution, guinea pigs presented a more frequent and broadly observed increase in disease score and corneal opacity, a phenomenon rarely seen in rabbits within the same timeframe. The effectiveness of 1% trifluridine was observed in mitigating ocular HSV-1 disease in both species, whereas oral acyclovir and valacyclovir were found to be detrimental and ineffective in guinea pigs but not in rabbits. Conclusions: This study demonstrates the potential suitability of guinea pigs as new models for ocular HSV-1 investigations, filling a critical preclinical void of models capable of showcasing spontaneous HSV reactivation in the eye. The observed similarities and differences in the reactions of guinea pigs and rabbits to HSV-1 infection and treatments provide crucial insights, laying the foundation for future studies on ocular HSV pathogenesis, latency, and improved treatment options.

Putative targeting by BX795 causes decrease in protein kinase C protein levels and inhibition of HSV1 infection.

Herpes simplex virus type-1 (HSV1) exploits cellular machinery for its own replicative advantage. Current treatment modalities against HSV1 cause toxicity and drug resistance issues. In the search for alternative forms of treatment, we have uncovered a small molecule, BX795, as a candidate drug with strong antiviral potential owing to its multitargeted mode of action. In this study, we show that in addition to a previously known mechanism of action, BX795 can directly interact with the proviral host factor protein kinase C (PKC) in silico. When administered to HSV1 or mock infected human corneal epithelial (HCE) cells, BX795 significantly reduces the protein level and perinuclear localization of proviral PKC-α and PKC-ζ isoforms. This activity closely mimics that of a known PKC inhibitor, Bisindolylmaleimide I (BIM I), which also inhibits viral replication. Taken together our studies demonstrate a previously unknown mechanism by which BX795 exerts its antiviral potential.

Aptamers in Virology-A Consolidated Review of the Most Recent Advancements in Diagnosis and Therapy.

The use of short oligonucleotide or peptide molecules as target-specific aptamers has recently garnered substantial attention in the field of the detection and treatment of viral infections. Based on their high affinity and high specificity to desired targets, their use is on the rise to replace antibodies for the detection of viruses and viral antigens. Furthermore, aptamers inhibit intracellular viral transcription and translation, in addition to restricting viral entry into host cells. This has opened up a plethora of new targets for the research and development of novel vaccines against viruses. Here, we discuss the advances made in aptamer technology for viral diagnosis and therapy in the past decade.

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

Herpes Simplex Virus Cell Entry Mechanisms: An Update.

Herpes simplex virus (HSV) can infect a broad host range and cause mild to life threating infections in humans. The surface glycoproteins of HSV are evolutionarily conserved and show an extraordinary ability to bind more than one receptor on the host cell surface. Following attachment, the virus fuses its lipid envelope with the host cell membrane and releases its nucleocapsid along with tegument proteins into the cytosol. With the help of tegument proteins and host cell factors, the nucleocapsid is then docked into the nuclear pore. The viral double stranded DNA is then released into the host cell's nucleus. Released viral DNA either replicates rapidly (more commonly in non-neuronal cells) or stays latent inside the nucleus (in sensory neurons). The fusion of the viral envelope with host cell membrane is a key step. Blocking this step can prevent entry of HSV into the host cell and the subsequent interactions that ultimately lead to production of viral progeny and cell death or latency. In this review, we have discussed viral entry mechanisms including the pH-independent as well as pH-dependent endocytic entry, cell to cell spread of HSV and use of viral glycoproteins as an antiviral target.

BX795 demonstrates potent antiviral benefits against herpes simplex Virus-1 infection of human cell lines.

Herpes simplex virus-1 (HSV-1) infection is known to cause skin blisters, keratitis as well as deadly cases of encephalitis in some situations. Only a few therapeutic modalities are available for this globally prevalent infection. Very recently, a small molecule BX795 was identified as an inhibitor of HSV-1 protein synthesis in an ocular model of infection. In order to demonstrate its broader antiviral benefits, this study was aimed at evaluating the antiviral efficacy, mode-of-action, and toxicity of BX795 against HSV-1 infection of three human cell lines: HeLa, HEK, and HCE. Several different assays, including cell survival analysis, imaging, plaque analysis, Immunoblotting, and qRT-PCR, were performed. In all cases, BX795 demonstrated low toxicity at therapeutic concentration and showed strong antiviral benefits. Quite interestingly, cell line-dependent differences in the mechanism of antiviral action and cytokine response to infection were seen upon BX795 treatment. Taken together, our results suggest that BX795 may exert its antiviral benefits via cell-line specific mechanisms.