Determination of the minimum infective dose of viral hemorrhagic septicemia virus (VHSV) in juvenile olive flounder, Paralichthys olivaceus using an immersion challenge model.

Viral hemorrhagic septicemia virus (VHSV) affects over 80 fish species, leading to viral hemorrhagic septicemia (VHS). Horizontal VHSV transmission is widely studied, with researchers utilizing various doses to establish infection models. Infected hosts shed the virus into the environment, elevating the risk of transmission to naïve fish within the same system. This study aimed to ascertain the minimum infective dose of VHSV in olive flounder (Paralichthys olivaceus). In olive flounder, the detection of VHSV within the kidney exhibited the highest infection rate on the third day among days 1, 3 and 5. Doses of 103.0 to 104.7 TCID50/ml were administered to juvenile olive flounder across three farms. Results showed resistance to infection below 103.4 TCID50/ml at 15 °C. While infection frequency varied by concentration, higher concentrations correlated with more infections. Nonetheless, viral copy numbers did not differ significantly among infected fish at varying concentrations. This study underscores the need for early VHSV management and contributes essential data for pathogenicity assessment and foundational knowledge.

Comparing machine learning and logistic regression for acute kidney injury prediction in trauma patients: A retrospective observational study at a single tertiary medical center.

Acute kidney injury (AKI) is common in patients with trauma and is associated with poor outcomes. Therefore, early prediction of AKI in patients with trauma is important for risk stratification and the provision of optimal intensive care unit treatment. This study aimed to compare 2 models, machine learning (ML) techniques and logistic regression, in predicting AKI in patients with trauma. We retrospectively reviewed the charts of 400 patients who sustained torso injuries between January 2016 and June 2020. Patients were included if they were aged > 15 years, admitted to the intensive care unit, survived for > 48 hours, had thoracic and/or abdominal injuries, had no end-stage renal disease, and had no missing data. AKI was defined in accordance with the Kidney Disease Improving Global Outcomes definition and staging system. The patients were divided into 2 groups: AKI (n = 78) and non-AKI (n = 322). We divided the original dataset into a training (80%) and a test set (20%), and the logistic regression with stepwise selection and ML (decision tree with hyperparameter optimization using grid search and cross-validation) was used to build a model for predicting AKI. The models established using the training dataset were evaluated using a confusion matrix receiver operating characteristic curve with the test dataset. We included 400 patients with torso injury, of whom 78 (19.5%) progressed to AKI. Age, intestinal injury, cumulative fluid balance within 24 hours, and the use of vasopressors were independent risk factors for AKI in the logistic regression model. In the ML model, vasopressors were the most important feature, followed by cumulative fluid balance within 24 hours and packed red blood cell transfusion within 4 hours. The accuracy score showed no differences between the 2 groups; however, the recall and F1 score were significantly higher in the ML model (.94 vs 56 and.75 vs 64, respectively). The ML model performed better than the logistic regression model in predicting AKI in patients with trauma. ML techniques can aid in risk stratification and the provision of optimal care.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection.

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFß. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

NLRC3 attenuates antiviral immunity and activates inflammasome responses in primary grouper brain cells following nervous necrosis virus infection.

NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.

Structure-activity relationship of leucyladenylate sulfamate analogues as leucyl-tRNA synthetase (LRS)-targeting inhibitors of Mammalian target of rapamycin complex 1 (mTORC1).

Leucyl-tRNA synthetase (LRS) plays an important role in amino acid-dependent mTORC1 signaling, which is known to be associated with cellular metabolism and proliferation. Therefore, LRS-targeting small molecules that can suppress mTORC1 activation may provide an alternative strategy to current anticancer therapy. In this work, we developed a library of leucyladenylate sulfate analogues by extensively modifying three different pharmacophoric regions comprising adenine, ribose and leucine. Several effective compounds were identified by cell-based mTORC1 activation assays and further tested for anticancer activity. The selected compounds mostly exhibited selective cytotoxicity toward five different cancer cell lines, supporting the hypothesis that the LRS-mediated mTORC1 pathway is a promising alternative target to current therapeutic approaches.

Discovery of novel leucyladenylate sulfamate surrogates as leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors.

According to recent studies, leucyl-tRNA synthetase (LRS) acts as a leucine sensor and modulates the activation of the mammalian target of rapamycin complex 1 (mTORC1) activation. Because overactive mTORC1 is associated with several diseases, including colon cancer, LRS-targeted mTORC1 inhibitors represent a potential option for anti-cancer therapy. In this work, we developed a series of simplified leucyladenylate sulfamate analogues that contain the N-(3-chloro-4-fluorophenyl)quinazolin-4-amine moiety to replace the adenine group. We identified several compounds with comparable activity to previously reported inhibitors and exhibited selective mTORC1 inhibition and anti-cancer activity. This study further supports the hypothesis that LRS is a promising target to modulate the mTORC1 pathway.

Discovery of simplified leucyladenylate sulfamates as novel leucyl-tRNA synthetase (LRS)-targeted mammalian target of rapamycin complex 1 (mTORC1) inhibitors.

Leucyl-tRNA synthetase (LRS) has been reported to be a possible mediator of intracellular amino acids signaling to mTORC1. Given that mTORC1 is associated with cell proliferation and tumorigenesis, the LRS-mediated mTORC1 pathway may offer an alternative strategy in anticancer therapy. In this study, we developed a series of simplified analogues of leucyladenylate sulfamate (1) as LRS-targeted mTORC1 inhibitors. We replaced the adenylate group with a N-(3,4-dimethoxybenzyl)benzenesulfonamide (2a) or a N-(2-phenoxyethyl)benzenesulfonamide groups (2b) that can maintain specific binding, but has more favorable physicochemical properties such as reduced polarity and asymmetric centers. Among these simplified analogues, compound 16 and its constrained analogue 22 effectively inhibited S6K phosphorylation in a dose-dependent manner and exhibited cancer cell specific cytotoxicity against six different types of cancer cells. This result supports that LRS is a viable target for novel anticancer therapy.

Discovery of Leucyladenylate Sulfamates as Novel Leucyl-tRNA Synthetase (LRS)-Targeted Mammalian Target of Rapamycin Complex 1 (mTORC1) Inhibitors.

Recent studies indicate that LRS may act as a leucine sensor for the mTORC1 pathway, potentially providing an alternative strategy to overcome rapamycin resistance in cancer treatments. In this study, we developed leucyladenylate sulfamate derivatives as LRS-targeted mTORC1 inhibitors. Compound 18 selectively inhibited LRS-mediated mTORC1 activation and exerted specific cytotoxicity against colon cancer cells with a hyperactive mTORC1, suggesting that 18 may offer a novel treatment option for human colorectal cancer.

Synthesis and biological evaluation of C-ring truncated deguelin derivatives as heat shock protein 90 (HSP90) inhibitors.

Based on the lead compound L-80 (compound 2), a potent heat shock protein 90 (HSP90) inhibitor, a series of C-ring truncated deguelin analogs were designed, synthesized and evaluated for Hypoxia Inducible Factor-1α (HIF-1α) inhibition as a primary screening method. Their structure-activity relationship was investigated in a systematic manner by varying the A/B ring, linker and D/E ring, respectively. Among the synthesized inhibitors, compound 5 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in human non-small cell lung carcinoma (H1299), with better activities than L-80. It also inhibited in vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells (HRMEC). The docking study of 5 showed a similar binding mode as L-80: it occupied the C-terminal ATP-binding pocket of HSP90, indicating that the anticancer and antiangiogenic activities of 5 were derived from HIF-1α destabilization by inhibiting the C-terminal ATP-binding site of hHSP90.

Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist.

We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected 18a (SKI2496) for further in vivo studies. Compound 18a exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of 18a revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that 18a exhibited highly potent and selective antagonistic activity toward the hGnRHRs along with favorable pharmacokinetic profiles, we believe that 18a may represent a promising candidate for an orally available hormonal therapy.

Discovery of N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea as a potent TRPV1 antagonistic template.

A series of homologous analogues of prototype antagonist 1 and its urea surrogate were investigated as hTRPV1 ligands. Through one-carbon elongation in the respective pharmacophoric regions, N-(3-fluoro-4-methylsulfonamidomethylphenyl)urea was identified as a novel and potent TRPV1 antagonistic template. Its representative compound 27 showed a potency comparable to that of lead compound 1. Docking analysis of compound 27 in our hTRPV1 homology model indicated that its binding mode was similar with that of 1S.

Discovery of (S)-4-isobutyloxazolidin-2-one as a novel leucyl-tRNA synthetase (LRS)-targeted mTORC1 inhibitor.

A series of leucinol analogs were investigated as leucyl-tRNA synthetase-targeted mTORC1 inhibitors. Among them, compound 5, (S)-4-isobutyloxazolidin-2-one, showed the most potent inhibition on the mTORC1 pathway in a concentration-dependent manner. Compound 5 inhibited downstream phosphorylation of mTORC1 by blocking leucine-sensing ability of LRS, without affecting the catalytic activity of LRS. In addition, compound 5 exhibited cytotoxicity against rapamycin-resistant colon cancer cells, suggesting that LRS has the potential to serve as a novel therapeutic target.

2-Sulfonamidopyridine C-region analogs of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-sulfonamidopyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamide were investigated as hTRPV1 ligands. Systematic modification on the 2-sulfonamido group provided highly potent TRPV1 antagonists. The N-benzyl phenylsulfonamide derivatives 12 and 23 in particular showed higher affinities than that of lead compound 1. Compound 12 exhibited strong analgesic activity in the formalin pain model. Docking analysis of its chiral S-form 12S in our hTRPV1 homology model indicated that its high affinity might arise from additional hydrophobic interactions not present in lead compound 1S.

Pyridine C-region analogs of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists.

A series of pyridine derivatives in the C-region of N-((6-trifluoromethyl-pyridin-3-yl)methyl) 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. The SAR analysis indicated that 6-difluorochloromethyl pyridine derivatives were the best surrogates of the C-region for previous leads. Among them, compound 31 showed excellent antagonism to capsaicin as well as to multiple hTRPV1 activators. It demonstrated strong analgesic activity in the formalin test in mice with full efficacy and it blocked capsaicin-induced hypothermia in vivo.

6,6-Fused heterocyclic ureas as highly potent TRPV1 antagonists.

A series of N-[{2-(4-methylpiperidin-1-yl)-6-(trifluoromethyl)-pyridin-3-yl}methyl] N'-(6,6-fused heterocyclic) ureas have been investigated as hTRPV1 antagonists. Among them, compound 15 showed highly potent TRPV1 antagonism to capsaicin, with Ki(ant)=0.2nM, as well as antagonism to other activators, and it was efficacious in a pain model. A docking study of 15 with our hTRPV1 homology model indicates that there is crucial hydrogen bonding between the ring nitrogen and the receptor, contributing to its potency.

Design and synthesis of protein kinase C epsilon selective diacylglycerol lactones (DAG-lactones).

DAG-lactones afford a synthetically accessible, high affinity platform for probing structure activity relationships at the C1 regulatory domain of protein kinase C (PKC). Given the central role of PKC isoforms in cellular signaling, along with their differential biological activities, a critical objective is the design of isoform selective ligands. Here, we report the synthesis of a series of DAG-lactones varying in their side chains, with a particular focus on linoleic acid derivatives. We evaluated their selectivity for PKC epsilon versus PKC alpha both under standard lipid conditions (100% phosphatidylserine, PS) as well as in the presence of a nuclear membrane mimetic lipid mixture (NML). We find that selectivity for PKC epsilon versus PKC alpha tended to be enhanced in the presence of the nuclear membrane mimetic lipid mixture and, for our lead compound, report a selectivity of 32-fold.

Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands.

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.

CM1 ligation initiates apoptosis in a caspase 8-dependent manner in Ramos cells and in a mitochondria-controlled manner in Raji cells.

Burkitt lymphoma (BL) is a tumor with the characteristics of germinal center B cells. We previously reported that the CM1 (centrocyte/-blast marker 1) molecule is expressed only in germinal center B cells, specifically, in a subpopulation of centroblasts and centrocytes. In the present study, we investigated the apoptosis induced by anti-CM1 in the Ramos and Raji human BL cell lines. The Ramos is protected from apoptosis by the crosslinking of sIgM and the calcium ionophore by the ligation of CD40 with anti-CD40 monoclonal antibodies (mAb) or soluble CD40 ligand (sCD40L). In this investigation on the effect of CM1 on apoptosis in BL cell lines, we found that cellular signaling by CM1 induces apoptosis and decreases cell viability, in BL cell lines cultured for 24 hours with protein-G agarose beads conjugated anti-CM1 mAb. Stimulation by CD40 ligated with sCD40L protected Raji cells from CM1-induced apoptosis, but did not protect Ramos cells. Furthermore, after anti-CM1 mAb stimulation, CD95 expression was upregulated and CD40 expression was unaltered or slightly decreased in Ramos cells, whereas CD95 was downregulated and CD40 was slightly upregulated in Raji cells. The engagement of CD40 by sCD40L enhanced CD95 expression, but the level of CM1 expression was unchanged in Ramos. However, sCD40L downregulated both CD95 and CM1 expression in Raji. In addition, the caspase-8 specific inhibitor blocked CM1-induced apoptosis in Ramos cells, but not in Raji cells. Increased mitochondrial membrane permeabilization was observed only in Raji cells. Moreover, the effector caspase inhibitor, z-DEVD, blocked CM1-mediated apoptosis in both cell lines. We found that CM1-induced apoptosis is achieved via different initiation pathways, which are cell-type dependent.