Unveiling the Antiviral Properties of Panduratin A through SARS-CoV-2 Infection Modeling in Cardiomyocytes.

Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC50) 10.09 µM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC50) 0.8-1.6 µM), suppressing infection at concentrations 10 times lower than its CC50. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications.

A Rhodamine-coumarin Triazole Conjugate as a Fluorescent Chemodosimeter for Cu(II) Detection and its Application in Live Cell Bioimaging.

A rhodamine-triazole fluorescent probe bearing a coumarin moiety RTC was synthesized using the Cu(I)-catalyzed click reaction. The rhodamine-triazole conjugate was highly selective to Cu2+ among other metal ions, including Ca2+, Co2+, Cu2+, Cd2+, Mg2+, Fe2+, Fe3+, Hg2+, Zn2+, Ni2+, Pd2+ and Pb2+ in physiological conditions. Upon the addition of Cu2+, the colorless RTC solution turned pink and exhibited a significant fluorescence emission centered at 578 nm. The binding of Cu2+ induced a hydrolysis reaction, leading to a release of the coumarin unit from the rhodamine probe, as confirmed by mass spectrometric data. From the fluorescence titration, the detection limit of RTC for Cu2+ was determined to be 21 nM (1.3 ppb). The sensor was responsive to Cu2+ in a wide pH range and successfully applied to monitor Cu2+ in HEK293T cells by confocal fluorescence imaging.

Bioassay-Guided Isolation and Identification of Antiplasmodial Compounds from the Stem Bark of Clausena excavata.

Clausena excavata is a medicinal plant widely distributed in Southeast Asia. It is used for a variety of indications, including to treat malaria. In our present study, a phytochemical study of the methanol extract from the stem bark of C. excavata led to the isolation of five pyranocoumarins, nordentatin (1: ), dentatin (2: ), kinocoumarin (3: ), clausarin (4: ), and clausenidin (5: ), and a coumarin, 8-hydroxy-3″,4″-dihydrocapnolactone-2',3'-diol (6: ). The isolation of compound 6: from C. excavata and the antiplasmodial activities against a multidrug-resistant K1 strain of Plasmodium falciparum of 1, 3: , and 5: were reported for the first time. Compounds 3: and 4: exhibited potent antiplasmodial activities with EC50 values of 1.10 and 0.58 µM, respectively, while 1: and 5: had EC50 values of 5.62 and 7.15 µM, respectively. A prenyl group attached to the C-3 or C-12 position on the pyranocoumarin ring probably plays an important role on the activity. A hydroxyl group at the C-10 position is also likely to enhance the activity.

Rational Design and Lead Optimisation of Potent Antimalarial Quinazolinediones and Their Cytotoxicity against MCF-7.

Quinazolinedione is one of the most outstanding heterocycles in medicinal chemistry thanks to its wide ranges of biological activities including antimalarial, anticancer, and anti-inflammatory. TCMDC-125133 containing a quinazolinedione pharmacophore displays promising antimalarial activity and low toxicity, as described in the GlaxoSmithKline (GSK) report. Herein, the design and synthesis of novel quinazolinedione derivatives is described on the basis of our previous work on the synthesis of TCMDC-125133, where low-cost chemicals and greener alternatives were used when possible. The initial SAR study focused on the replacement of the valine linker moiety; according to the in silico prediction using SwissADME, concise four-step syntheses toward compounds 4-10 were developed. The in-house synthesized compounds 4-10 were assayed for antimalarial activity against P. falciparum 3D7, and the result revealed that only the compound 2 containing a valine linker was tolerated. Another round of lead optimization focused on the replacement of the m-anisidine moiety in compound 2. A library of 12 derivatives was prepared, and the antimalarial assay showed that potent antimalarial activity could be maintained by replacing the methoxy group in the meta position of the phenyl side chain with a fluorine or chlorine atom (21: IC50 = 36 ± 5 nM, 24: IC50 = 22 ± 5 nM). Further lead optimization is underway to enhance the antimalarial activity of this class of compound. The compounds included in the study possess little to no antiproliferative activity against MCF-7 cells.

A Rhodamine-based Fluorescent Chemodosimeter for Au3+ in Aqueous Solution and Living Cells.

A highly selective rhodamine hydrazide-based fluorescent chemosensor for Au3+ detection was developed. The aqueous solution of rhodamine N-hydroxysemicarbazide (RHS), in the presence of Au3+, exhibited a significant 55-fold turn-on fluorescence response at 591 nm and a colorimetric change from colorless to pink. Other interested ions including Li+, Na+, K+, Cs+, Mg2+, Ca2+, Ba2+, Pb2+, Mn2+, Co2+, Ni2+, Ag+, Cd2+, Cu2+, Hg2+, Zn2+, Sn2+, Fe2+, Fe3+, Cr3+, Ce3+ did not induce any distinct color/spectral changes. The irreversible detection mechanism occurred via Au3+-promoted 5-exo-trig ring closure to yield 1,3,4-oxadiazole-2-one product. The RHS probe is non-responsive to other biologically relevant metal ions and the limit of detection for Au3+ was calculated to be 0.5 µM with a linear range of 0 to 90 µM. Fluorescence bioimaging of Au3+ in HepG2 cells was also successfully demonstrated.

Anti-SARS-CoV-2 Activity of Andrographis paniculata Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of Andrographis paniculata extract and its major component, andrographolide. SARS-CoV-2 at 25TCID50 was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of A. paniculata and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC50 of 0.036 µg/mL and 0.034 µM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC50 of >100 µg/mL for A. paniculata extract and 13.2-81.5 µM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of A. paniculata and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.

A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax.

BACKGROUND: Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay. METHODS: A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay. RESULTS: imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening. CONCLUSIONS: imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.

A rhodamine-triazole fluorescent chemodosimeter for Cu2+ detection and its application in bioimaging.

A rhodamine-based fluorescent chemodosimeter rhodamine hydrazide-triazole (RHT) tethered with a triazole moiety was developed for Cu2+ detection. In aqueous medium, the RHT probe exhibited high selectivity and sensitivity toward Cu2+ among other metal ions. The addition of Cu2+ triggered a fluorescence emission of RHT by 384-fold (Φ = 0.33) based on a ring-opening process and a subsequent hydrolysis reaction. Moreover, RHT also showed a selective colorimetric response toward Cu2+ from colorless solution to pink, readily observed with the naked eye. The limit of detection of RHT for Cu2+ was calculated to be 1 nM (0.06 ppb). RHT was successfully demonstrated to detect Cu2+ in Chang liver cells by confocal fluorescence microscopy.

Investigating the impact of botulinum toxin type a on the migration of normal human dermal fibroblasts: An in vitro wound healing assay.

BACKGROUND: Botulinum toxin A (BoNT-A) is widely utilized in the management of hypertrophic and keloid scars. One proposed mechanism for scar prevention involves the inhibition of fibroblast migration in scars by BoNT-A. However, the data regarding the effect of BoNT-A on the migration of normal human dermal fibroblasts (NHDF) is limited. OBJECTIVES: The aim of this study was to investigate the inhibitory effect of different types and dilutions of BoNT-A on the migration of NHDF. METHODS: In vitro scratch wound assay, NHDF cells were cultured, incubated, and subjected to scratching using a sterile tip. Subsequently, the scratched NHDF monolayer was treated with different types of BoNT-A, including onabotulinumtoxinA (ONA), incobotulinumtoxinA (INCO), prabotulinumtoxinA (PRABO), or letibotulinumtoxinA (LETI), at varying concentrations of 10, 20, 25, 40, 50, and 100 units/milliliter (U/mL). Additionally, abobotulinumtoxinA (ABO) was administered at concentrations of 33, 50, 66, 71, 100, 150, 300, and 500 U/mL. Normal saline solution (NSS) served as a negative control. The extent of NHDF migration was evaluated by comparing each dilution of BoNT-A with the controls using high-content imaging at the 48-h time point. Furthermore, the viability of the of NHDF was assessed. RESULTS: The concentrations of 25, 40, and 50 U/mL of ONA (p < 0.001) and 25 U/mL of LETI (p < 0.05) demonstrated significantly inhibited NHDF migration in comparison to the control group. Conversely, all dilutions of PRABO, INCO, and ABO exhibited comparable NHDF migration to that of the control group. Regarding NHDF viability, no significant decrease was observed across any of the BoNT-A types and dilutions. CONCLUSION: Different types and dilutions of BoNT-A demonstrated variable inhibitory effects on NHDF migration in vitro. The selection of BoNT-A formulation may significantly impact the clinical outcome of scar prevention related to fibroblast migration.

GDNF and cAMP significantly enhance in vitro blood-brain barrier integrity in a humanized tricellular transwell model.

Blood-brain barrier (BBB) is a crucial membrane safeguarding neural tissue by controlling the molecular exchange between blood and the brain. However, assessing BBB permeability presents challenges for central nervous system (CNS) drug development. In vitro studies of BBB-permeable agents before animal testing are essential to mitigate failures. Improved in vitro models are needed to mimic physiologically relevant BBB integrity. Here, we established an in vitro human-derived triculture BBB model, coculturing hCMEC/D3 with primary astrocytes and pericytes in a transwell format. This study found that the triculture BBB model exhibited significantly higher paracellular tightness (TEER 147.6 ± 6.5 Ω × cm2) than its monoculture counterpart (106.3 ± 1.0 Ω × cm2). Additionally, BBB permeability in the triculture model was significantly lower. While GDNF and cAMP have been shown to promote BBB integrity in monoculture models, their effect in our model was previously unreported. Our study demonstrates that both GDNF and cAMP increased TEER values (around 200 Ω × cm2 for each; 237.6 ± 17.7 Ω × cm2 for co-treatment) compared to untreated control, and decreased BBB permeability, mediated by increased claudin-5 expression. In summary, this humanized triculture BBB model, enhanced by GDNF and cAMP, offers an alternative for exploring in vitro drug penetration into the human brain.

A highly sensitive and selective rhodamine-semicarbazide based fluorescent sensor for Cu2+ detection in real water samples and fluorescence bioimaging in HepG2 cells.

A colorimetric and fluorescent sensor, selective for Cu2+ ions, was synthesized in two steps using a rhodamine-based compound attached to the semicarbazide-picolylamine moiety (RBP). Spectroscopic measurements, including UV-Vis absorption and fluorescence emission, were conducted in the semi-aqueous medium containing acetonitrile/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, denoted as MeCN/HEPES buffer (2:8, v/v, pH 7.0). The sensor exhibited high selectivity towards Cu2+ ions compared to other cations and demonstrated remarkable sensitivity towards Cu2+ ions, with a limit of detection at the nanomolar level. The calculated transitions indicated a 1:1 stoichiometric binding of RBP to Cu2+ ions based on a 4-coordination mode involving additional chelation in the semi-aqueous medium. The sensing mechanism for the detection of Cu2+ ions was investigated using high-resolution mass spectroscopy. The sensor could be employed as a real-time chemosensor for monitoring Cu2+ ions. Furthermore, the sensor has the potential for utilization in the detection of Cu2+ ions in actual water samples with the high precision and accuracy, as indicated by the small relative standard derivation values. The 50th percentile cytotoxicity concentration of RBP was found to be 22.92 µM. Additionally, the fluorescence bioimaging capability of RBP was demonstrated for the detection of Cu2+ ions in human hepatocellular carcinoma (HepG2) cells.

Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

Cleistanthin A derivative disrupts autophagy and suppresses head and neck squamous cell carcinoma progression via targeted vacuolar ATPase.

Head and neck squamous cell carcinoma (HNSCC) present a significant challenge due to its heterogeneity and limited treatment options, often resulting in severe side effects and poor survival rates with conventional chemoradiotherapy. Here, we investigated the anticancer activity of halogenated benzoate derivatives of cleistanthin A, ECDD-S16 and ECDD-S18, in HNSCC cells. Our findings revealed that ECDD-S18 exhibited remarkable cytotoxicity, surpassing that of cisplatin with minimal impact on normal and cisplatin-sensitive cells. Notably, ECDD-S18 induced apoptosis in a dose-dependent manner and effectively targeted vacuolar ATPase (V-ATPase), impairing lysosomal acidification. Intriguingly, ECDD-S18 inhibited autophagic flux, as evidenced by increased autophagosome but decreased autolysosome formation. Furthermore, proteomic analysis demonstrated downregulation of cathepsin D (CTSD), the lysosomal protease in ECDD-S18-treated HNSCC cells, concurrent with suppressed cell migration. ECDD-S18 also decreased expression of mesenchymal markers, suggesting inhibition of epithelial-mesenchymal transition (EMT). Importantly, cotreatment with ECDD-S18 and cisplatin enhanced the reduction in cell viability. Collectively, our results indicated that the anticancer activity of ECDD-S18 partly stems from its ability to disrupt lysosomal acidification and inhibit autophagy via targeted inhibition of V-ATPase. These findings underscore the therapeutic promise of ECDD-S18 in HNSCC treatment, either alone or in combination with existing drugs, while mitigating toxicity to normal cells.

BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain.

Autophagy induction by starvation has been shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of the Mycobacterium tuberculosis reference strain H37Rv. In contrast to H37Rv, our previous study showed that strains belonging to the notorious M. tuberculosis Beijing genotype could evade autophagic elimination. Our recent RNA-Seq analysis also discovered that the autophagy-resistant M. tuberculosis Beijing strain (BJN) evaded autophagic control by upregulating the expression of Kxd1, a BORC complex component, and Plekhm2, both of which function in lysosome positioning towards the cell periphery in host macrophages, thereby suppressing enhanced lysosomal delivery to its phagosome and sparing the BJN from elimination as a result. In this work, we further characterised the other specific components of the BORC complex, BORC5-8, and Kinesin proteins in autophagy resistance by the BJN. Depletion of BORCS5-8 and Kinesin-1, but not Kinesin-3, reverted autophagy avoidance by the BJN, resulting in increased lysosomal delivery to the BJN phagosomes. In addition, the augmented lysosome relocation towards the perinuclear region could now be observed in the BJN-infected host cells depleted in BORCS5-8 and Kinesin-1 expressions. Taken together, the data uncovered new roles for BORCS5-8 and Kinesin-1 in autophagy evasion by the BJN.

Novel Potent Autophagy Inhibitor Ka-003 Inhibits Dengue Virus Replication.

Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.

Upregulated LAMA3 modulates proliferation, adhesion, migration and epithelial­to­mesenchymal transition of cholangiocarcinoma cells.

A poor outcome for cholangiocarcinoma (CCA) patients is still a clinical challenge. CCA is typically recognized by the desmoplastic nature, which accounts for its malignancy. Among various extracellular matrix proteins, laminin is the most potent inducer for CCA migration. Herein, we accessed the expression profiles of laminin gene family and explored the significance of the key laminin subunit on CCA aggressiveness. Of all 11 laminin genes, LAMA3, LAMA5, LAMB3 and LAMC2 were concordantly upregulated based on the analysis of multiple public transcriptomic datasets and also overexpressed in Thai CCA cell lines and patient tissues in which LAMA3A upregulated in the highest frequency (97%) of the cases. Differential expression genes (DEGs) analysis of low and high laminin signature groups revealed LAMA3 as the sole common DEG in all investigated datasets. Restratifying CCA samples according to LAMA3 expression indicated the association of LAMA3 in the focal adhesion pathway. Silencing LAMA3 revealed that it plays important roles in CCA cell proliferation, adhesion, migration and epithelial-to-mesenchymal transition. Taken together, this research signifies the roles of dysregulated ECM homeostasis in CCA malignancy and highlights, for the first time, the potential usage of LAMA3 as the diagnostic biomarker and the therapeutic target to tackle the CCA stromal.

Pharmacological Activities of Fingerroot Extract and Its Phytoconstituents Against SARS-CoV-2 Infection in Golden Syrian Hamsters.

Background: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems. Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo. Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A. Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals. Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development.

Host cell transcriptomic response to the multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

The upsurge of multidrug-resistant infections has rendered tuberculosis the principal cause of death among infectious diseases. A clonal outbreak multidrug-resistant triggering strain of Mycobacterium tuberculosis was identified in Kanchanaburi Province, labelled "MKR superspreader," which was found to subsequently spread to other regions, as revealed by prior epidemiological reports in Thailand. Herein, we showed that the MKR displayed a higher growth rate upon infection into host macrophages in comparison with the H37Rv reference strain. To further elucidate MKR's biology, we utilized RNA-Seq and differential gene expression analyses to identify host factors involved in the intracellular viability of the MKR. A set of host genes function in the cellular response to lipid pathway was found to be uniquely up-regulated in host macrophages infected with the MKR, but not those infected with H37Rv. Within this set of genes, the IL-36 cytokines which regulate host cell cholesterol metabolism and resistance against mycobacteria attracted our interest, as our previous study revealed that the MKR elevated genes associated with cholesterol breakdown during its growth inside host macrophages. Indeed, when comparing macrophages infected with the MKR to H37Rv-infected cells, our RNA-Seq data showed that the expression ratio of IL-36RN, the negative regulator of the IL-36 pathway, to that of IL-36G was greater in macrophages infected with the MKR. Furthermore, the MKR's intracellular survival and increased intracellular cholesterol level in the MKR-infected macrophages were diminished with decreased IL-36RN expression. Overall, our results indicated that IL-36RN could serve as a new target against this emerging multidrug-resistant M. tuberculosis strain.

The autophagy-resistant Mycobacterium tuberculosis Beijing strain upregulates KatG to evade starvation-induced autophagic restriction.

Mycobacterium tuberculosis utilizes several mechanisms to block phagosome-lysosome fusion to evade host cell restriction. However, induction of host cell autophagy by starvation was shown to overcome this block, resulting in enhanced lysosomal delivery to mycobacterial phagosomes and the killing of the M. tuberculosis reference strain H37Rv. Nevertheless, our previous studies found that strains belonging to the M. tuberculosis Beijing genotype can resist starvation-induced autophagic elimination, though the mycobacterial factors involved remain unclear. In this study, we showed that KatG expression is upregulated in the autophagy-resistant M. tuberculosis Beijing strain (BJN) during autophagy induction by the starvation of host macrophages, while such increase was not observed in the H37Rv. KatG depletion using the CRISPR-dCas9 interference system in the BJN resulted in increased lysosomal delivery to its phagosome and decreased its survival upon autophagy induction by starvation. As KatG functions by catabolizing ROS, we determined the source of ROS contributing to the starvation-induced autophagic elimination of mycobacteria. Using siRNA-mediated knockdown, we found that Superoxide dismutase 2, which generates mitochondrial ROS but not NADPH oxidase 2, is important for the starvation-induced lysosomal delivery to mycobacterial phagosomes. Taken together, these findings showed that KatG is vital for the BJN to evade starvation-induced autophagic restriction.

Development of a Cell-Based Assay for Identifying KCa3.1 Inhibitors Using Intestinal Epithelial Cell Lines.

Inhibition of the KCa3.1 potassium channel has therapeutic potential in a variety of human diseases, including inflammation-associated disorders and cancers. However, KCa3.1 inhibitors with high therapeutic promise are currently not available. This study aimed to establish a screening assay for identifying inhibitors of KCa3.1 in native cells and from library compounds derived from natural products in Thailand. The screening platform was successfully developed based on a thallium flux assay in intestinal epithelial (T84) cells with a Z' factor of 0.52. The screening of 1352 compounds and functional validation using electrophysiological analyses identified 8 compounds as novel KCa3.1 inhibitors with IC50 values ranging from 0.14 to 6.57 µM. These results indicate that the assay developed is of excellent quality for high-throughput screening and capable of identifying KCa3.1 inhibitors. This assay may be useful in identifying novel KCa3.1 inhibitors that may have therapeutic potential for inflammation-associated disorders and cancers.