Kynurenine 3-monooxygenase limits de novo NAD+ synthesis through dietary tryptophan in renal proximal tubule epithelial cell models.

Nicotinamide adenine dinucleotide (NAD+) is a pivotal coenzyme, essential for cellular reactions, metabolism, and mitochondrial function. Depletion of kidney NAD+ levels and reduced de novo NAD+ synthesis through the tryptophan-kynurenine pathway are linked to acute kidney injury (AKI), whereas augmenting NAD+ shows promise in reducing AKI. We investigated de novo NAD+ biosynthesis using in vitro, ex vivo, and in vivo models to understand its role in AKI. Two-dimensional (2-D) cultures of human primary renal proximal tubule epithelial cells (RPTECs) and HK-2 cells showed limited de novo NAD+ synthesis, likely due to low pathway enzyme gene expression. Using three-dimensional (3-D) spheroid culture model improved the expression of tubular-specific markers and enzymes involved in de novo NAD+ synthesis. However, de novo NAD+ synthesis remained elusive in the 3-D spheroid culture, regardless of injury conditions. Further investigation revealed that 3-D cultured cells could not metabolize tryptophan (Trp) beyond kynurenine (KYN). Intriguingly, supplementation of 3-hydroxyanthranilic acid into RPTEC spheroids was readily incorporated into NAD+. In a human precision-cut kidney slice (PCKS) ex vivo model, de novo NAD+ synthesis was limited due to substantially downregulated kynurenine 3-monooxygenase (KMO), which is responsible for KYN to 3-hydroxykynurenine conversion. KMO overexpression in RPTEC 3-D spheroids successfully reinstated de novo NAD+ synthesis from Trp. In addition, in vivo study demonstrated that de novo NAD+ synthesis is intact in the kidney of the healthy adult mice. Our findings highlight disrupted tryptophan-kynurenine NAD+ synthesis in in vitro cellular models and an ex vivo kidney model, primarily attributed to KMO downregulation.NEW & NOTEWORTHY Nicotinamide adenine dinucleotide (NAD+) is essential in regulating mitochondrial function. Reduced NAD+ synthesis through the de novo pathway is associated with acute kidney injury (AKI). Our study reveals a disruption in de novo NAD+ synthesis in proximal tubular models, but not in vivo, attributed to downregulation of enzyme kynurenine 3-monooxygenase (KMO). These findings highlight a crucial role of KMO in governing de novo NAD+ biosynthesis within the kidney, shedding light on potential AKI interventions.

Targeting Pan-ETS Factors Inhibits Melanoma Progression.

The failure of once promising target-specific therapeutic strategies often arises from redundancies in gene expression pathways. Even with new melanoma treatments, many patients are not responsive or develop resistance, leading to disease progression in terms of growth and metastasis. We previously discovered that the transcription factors ETS1 and PAX3 drive melanoma growth and metastasis by promoting the expression of the MET receptor. Here, we find that there are multiple ETS family members expressed in melanoma and that these factors have redundant functions. The small molecule YK-4-279, initially developed to target the ETS gene-containing translocation product EWS-FLI1, significantly inhibited cellular growth, invasion, and ETS factor function in melanoma cell lines and a clinically relevant transgenic mouse model, BrafCA;Tyr-CreERT2;Ptenf/f. One of the antitumor effects of YK-4-279 in melanoma is achieved via interference of multiple ETS family members with PAX3 and the expression of the PAX3-ETS downstream gene MET. Expression of exogenous MET provided partial rescue of the effects of YK-4-279, further supporting that MET loss is a significant contributor to the antitumor effects of the drug. This is the first study identifying multiple overlapping functions of the ETS family promoting melanoma. In addition, targeting all factors, rather than individual members, demonstrated impactful deleterious consequences in melanoma progression. Given that multiple ETS factors are known to have oncogenic functions in other malignancies, these findings have a high therapeutic impact. SIGNIFICANCE: These findings identify YK-4-279 as a promising therapeutic agent against melanoma by targeting multiple ETS family members and blocking their ability to act as transcription factors.

YK-4-279 Attenuates Progression of Pre-Existing Pigmented Lesions to Nodular Melanoma in a Mouse Model.

More options are needed for the effective treatment of melanoma. In a previous study, we discovered the small molecule drug YK-4-279 almost completely inhibited tumor progression in the BrafCA;Tyr-CreERT2;Ptenflox/flox transgenic mouse model. YK-4-279 had no effect on tumor initiation but blocked progression of invasive melanoma. Our current study was designed as a treatment model, where YK-4-279 was administered during pigmented lesion formation. The study design included the use of three groups: (1) a control group that received only DMSO without a drug (MOCK), (2) mice following our prior studies with YK-4-279 administered at the time of tumor induction (YK-4-279), and (3) mice treated during tumor initiation (YK-4-279 delay). While the MOCK mice had progression of tumors, both YK-4-279 and YK-4-279 delay groups had a significant block or delay of progression. The majority of mice in the YK-4-279 groups had a block of progression, while the YK-4-279 delay group had either a partial block (60% in male mice or 29% in females) or a delay in disease progression in females (28 days in controls to 50 days in YK-4-279 delay group). Here, we demonstrate that YK-4-279 has a significant impact on blocking or delaying tumor progression in a pre-clinical treatment model of melanoma.

Papilloma-pseudovirus eradicates intestinal tumours and triples the lifespan of ApcMin/+ mice.

Inducing tumour-specific adaptive immunity, such as cytotoxic T lymphocyte (CTL) response, can result in promising antitumour effect against several human malignancies, especially in combination with immune checkpoint blockade strategies. However, little is known whether activation of innate immunity can lead to direct tumoricidal effect. Here, we develop a papilloma pseudovirus-based oral immunotherapeutic approach that shows strong tumoricidal effects in the gut, resulting in an almost tripled lifespan of ApcMin/+ mice (an animal model of human intestinal tumorigenesis). Mechanistically, these pseudoviruses activate the NLRP3 and AIM2 inflammasomes, leading to caspase-1-mediated tumour regression that is dependent on neither cytotoxic T lymphocytes nor humoral immune response. Blocking caspase-1 activation abrogated the therapeutic effects of the pseudoviruses. Thus, targeting innate immune sensors in tumours by the pseudoviruses might represent a strategy to treat intestinal tumours.

Molecular evolution of emerging Banna virus.

Banna virus (BAV) is an emerging pathogen that causes human viral encephalitis and has been isolated from types of blood-sucking insects and mammals in Asia. However, there are no reported systematic studies that describe the origin and evolution of BAV. Here, a phylogenetic analysis of BAVs isolated from a variety of potential vectors and vertebrate hosts worldwide revealed that BAVs emerged in the beginning of the 20th century and do not exhibit a species barrier. The mean substitution rate of BAVs was 2.467×10-2substitution/site/year (95% HPD, 1.093×10-3 to 5.628×10-2). The lineage is mainly composed of BAVs from high-latitude regions, which are the most recently emerged viruses with significantly higher substitution rates compared with the lineage comprised of the isolates from middle or low-latitude regions. The genetic differences between BAV strains are positively correlated with the geographic distribution. Strains from the same latitude regions are almost 100% identical, whereas the differences between strains from long distance regions with different latitudes could be >60%. Our results demonstrate that BAV is an emerging virus at a stage that involves rapid evolution and has great potential for introduction into non-endemic areas. Thus, enhanced surveillance of BAV is highly recommended worldwide.

Protection of retinal function by sulforaphane following retinal ischemic injury.

Sulforaphane, a precursor of glucosinolate in cruciferous vegetables such as broccoli and cauliflower, has been shown to protect brain ischemic injury. In this study, we examined the effect of systemic administration of sulforaphane on retinal ischemic reperfusion injury. Intraocular pressure was elevated in two groups of C57BL/6 mice (n = 8 per group) for 45 min to induce retinal ischemic reperfusion injury. Following retinal ischemic reperfusion injury, vehicle (1% DMSO saline) or sulforaphane (25 mg/kg/day) was administered intraperitoneally daily for 5 days. Scotopic electroretinography (ERG) was used to quantify retinal function prior to and one-week after retinal ischemic insult. Retinal morphology was examined one week after ischemic insult. Following ischemic reperfusion injury, ERG a- and b-wave amplitudes were significantly reduced in the control mice. Sulforaphane treatment significantly attenuated ischemic-induced loss of retinal function as compared to vehicle treated mice. In vehicle treated mice, ischemic reperfusion injury produced marked thinning of the inner retinal layers, but the thinning of the inner retinal layers appeared significantly less with sulforaphane treatment. Thus, sulforaphane may be beneficial in the treatment of retinal disorders with ischemic reperfusion injury.

Effects of activated omental cells on rat limbal corneal alkali injury.

Omental cells (OCs) are shown to help wound healing. The purpose of this study is to investigate if OCs improve cornea repair after alkali injury by subconjunctival injection of activated OCs in rats. Forty eight hours after limbal corneal alkali injury, fresh isolated OCs were injected subconjunctivally into the recipient rat's eye. Prior to the injury and at 0, 4 and 8 days after injury, the eyes were examined using slit lamp biomicroscopy. Corneal opacification and corneal neovascularization were graded in a masked fashion. The inflammatory response to the injury was evaluated by counting neutrophil cell numbers in the cornea under microscope. There was no significant difference in corneal opacification between the control and OCs treatment groups; however, the corneal neovascularization was significantly less in the eyes treated with OCs as compared to the controls. Also OCs treatment markedly decreased neutrophil infiltration after corneal-limbal alkali injury. Our results suggest that OCs may have a beneficial role in corneal healing after limbal corneal alkali injury by suppressing inflammatory cell infiltrates and corneal neovascularization.

Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies.

Two conserved epitopes, located in the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41, are recognized by two HIV-1 broadly neutralizing antibodies 2F5 and 4E10, and are promising targets for vaccine design in efforts to elicit anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 or 4E10 epitope or the MPER domain into D-E loop of BPV L1 respectively. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting single epitopes. Oral immunization of mice with the chimeric VLPs displaying the 2F5 epitope or MPER domain elicited epitope-specific serum IgGs and mucosal secretory IgAs. The induced antibodies specifically recognized the native conformation of MPER in the context of HIV-1 envelope protein. The antibodies induced by chimeric VLPs presenting MPER domain are able to partially neutralize HIV-1 viruses from clade B and clade C.

Transient receptor potential melastatin 2: a novel target for treatment of gout.

Gout is an ancient autoinflammatory disease that affects millions of people worldwide. It is characterized by unbearable recurrent pain due to the massive local inflammation caused by the metabolic product, monosodium urate crystals. Although conventional therapies for gout can reduce the pain in patients, the severe undesirable side effects require the urgent need for novel therapies that can more specifically target gout-associated inflammatory pathways. Recent scientific advance on the mechanistic study of gout-associated inflammation is discussed and the potential of targeting the transient receptor potential melastatin 2 is highlighted as a novel therapeutic approach for the treatment of gout.

TRPM2 links oxidative stress to NLRP3 inflammasome activation.

Exposure to particulate crystals can induce oxidative stress in phagocytes, which triggers NLRP3 inflammasome-mediated interleukin-1ß secretion to initiate undesirable inflammatory responses that are associated with both autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species have a central role in NLRP3 inflammasome activation, how reactive oxygen species signal assembly of the NLRP3 inflammasome remains elusive. Here, we identify liposomes as novel activators of the NLRP3 inflammasome and further demonstrate that liposome-induced inflammasome activation also requires mitochondrial reactive oxygen species. Moreover, we find that stimulation with liposomes/crystals induced reactive oxygen species-dependent calcium influx via the TRPM2 channel and that macrophages deficient in TRPM2 display drastically impaired NLRP3 inflammasome activation and interleukin-1ß secretion. Consistently, Trpm2(-/-) mice are resistant to crystal-/liposome-induced interleukin-1ß-mediated peritonitis in vivo. Together, these results identify TRPM2 as a key factor that links oxidative stress to the NLRP3 inflammasome activation. Therefore, targeting TRPM2 may be effective for the treatment of NLRP3 inflammasome-associated inflammatory disorders.

Neuroprotective effect of resveratrol prophylaxis on experimental retinal ischemic injury.

The purpose of the present study was to investigate whether systemically administered resveratrol can protect against acute retinal ischemic reperfusion injury. Two groups of adult male Sprague Dawley rats (n = 6 per group) were used for this study. Resveratrol (30 mg/kg) or an equal volume of vehicle (30% Solutol HS 15 in 0.9% saline) was administered daily for 5 days via intraperitoneal injection. On the third day of treatment, retinal ischemic injury was induced by elevation of intraocular pressure for 45 min. Prior to resveratrol administration and one-week following ischemic insult, retinal function was measured by scotopic electroretinography (ERG). Retinas were harvested and morphologically analyzed one week after ischemic insult. ERG a- and b-wave amplitudes were significantly reduced following ischemic reperfusion injury. Resveratrol treatment attenuated ischemic-induced loss of retinal function. In control vehicle-treated rats, ischemic reperfusion injury elicited marked thinning of inner retinal layers. Resveratrol prophylactic treatment reduced ischemia-mediated thinning of the whole retina and in particular the inner retinal layers. Therefore, resveratrol may have therapeutic value for the management of retinal ischemic disorders.

Genomic characterization of a novel virus of the family Tymoviridae isolated from mosquitoes.

BACKGROUND: The family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV. METHODS AND RESULTS: The CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called "tymobox" or "marafibox", the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus. CONCLUSIONS: The CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.

Emergence of genotype I of Japanese encephalitis virus as the dominant genotype in Asia.

Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.

Identification and isolation of Genotype-I Japanese encephalitis virus from encephalitis patients.

Historically, Japanese Encephalitis virus (JEV) genotype III (GIII) has been responsible for human diseases. In recent years, JEV genotype I (GI) has been isolated from mosquitoes collected in numerous countries, but has not been isolated from patients with encephalitis. In this study, we report recovery of JEV GI live virus and identification of JEV GI RNA from cerebrospinal fluid (CSF) of encephalitis patients in JE endemic areas of China. Whole-genome sequencing and molecular phylogenetic analysis of the JEV isolate from the CSF samples was performed. The isolate in this study is highly similar to other JEV GI strains which isolated from mosquitoes at both the nucleotide and deduced amino acid levels. Phylogenetic analysis based on the genomic sequence showed that the isolate belongs to JEV GI, which is consistent with the phylogenetic analysis based on the pre-membrane (PrM) and Glycoprotein genes. As a conclusion, this is the first time to isolate JEV GI strain from CSF samples of encephalitis patients, so continuous survey and evaluate the infectivity and pathogenecity of JEV GI strains are necessary, especially for the JEV GI strains from encephalitis patients. With respect to the latter, because all current JEV vaccines (live and inactivated are derived from JEV GIII strains, future studies should be aimed at investigating and monitoring cross-protection of the human JEV GI isolates against widely used JEV vaccines.

Isolation and full-length sequence analysis of Armigeres subalbatus totivirus, the first totivirus isolate from mosquitoes representing a proposed novel genus (Artivirus) of the family Totiviridae.

During an investigation of arboviruses in China, a novel dsRNA virus was isolated from adult female Armigeres subalbatus. Full genome sequence analysis showed the virus to be related to members of the family Totiviridae, and was therefore named 'Armigeres subalbatus totivirus' (AsTV). Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 40 nm. The AsTV genome is 7510 bp in length, with two ORFs. ORF1 (4443 nt) encodes the coat-protein and a dsRNA-binding domain (which may be involved in the evasion of 'gene silencing'), while ORF2 (2286 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). The AsTV coat protein shows a higher level of amino acid identity with Drosophila totivirus (DTV, 52 %) than with infectious myonecrosis virus (IMNV, 29 %). Similarly, the RdRp shows higher identity levels with DTV (51 %) than with IMNV (44 %). Identity levels to other members of the family Totiviridae, in either the coat protein or the RdRp, ranged from 6 to 11 %. Based on a recent reassessment of the coding strategy used by IMNV, we suggest that an AsTV coat-RdRp fusion protein could be synthesized via a -1 frameshift. Elements favouring -1 frameshift such as 'slippery heptamers' and pseudonkots, were identified in the AsTV, DTV and IMNV genomes. AsTV was shown to grow in both mosquito and mammalian cells, suggesting that it is an arbovirus that can infect mammals.

Banna virus, China, 1987-2007.

Banna viruses (BAVs) have been isolated from pigs, cattle, ticks, mosquitoes, and human encephalitis patients. We isolated and analyzed 20 BAVs newly isolated in China; this finding extends the distribution of BAVs from tropical zone to north temperate climates and demonstrate regional variations in BAV phylogeny and mosquito species possibly involved in BAV transmission.