CAMSAP3-mediated regulation of HMGB1 acetylation and subcellular localization in lung cancer cells: Implications for cell death modulation.

BACKGROUND: Deregulation of cell death is a common characteristic of cancer, and resistance to this process often occurs in lung cancer. Understanding the molecular mechanisms underlying an aberrant cell death is important. Recent studies have emphasized the involvement of calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) in lung cancer aggressiveness, its influence on cell death regulation remains largely unexplored. METHODS: CAMSAP3 was knockout in lung cancer cells using CRISPR-Cas9 system. Cell death and autophagy were evaluated using MTT and autophagic detection assays. Protein interactions were performed by proteomic analysis and immunoprecipitation. Protein expressions and their cytoplasmic localization were analyzed through immunoblotting and immunofluorescence techniques. RESULTS: This study reveals a significant correlation between low CAMSAP3 expression and poor overall survival rates in lung cancer patients. Proteomic analysis identified high mobility group box 1 (HMGB1) as a candidate interacting protein involved in the regulation of cell death. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases (HDACs) resulted in increased HMGB1 acetylation and its translocation to the cytoplasm and secretion, thereby inducing autophagic cell death. However, this process was diminished in CAMSAP3 knockout lung cancer cells. Mechanistically, immunoprecipitation indicated an interaction between CAMSAP3 and HMGB1, particularly with its acetylated form, in which this complex was elevated in the presence of TSA. CONCLUSIONS: CAMSAP3 is prerequisite for TSA-mediated autophagic cell death by interacting with cytoplasmic acetylated HMGB1 and enhancing its release. SIGNIFICANT: This finding provides molecular insights into the role of CAMSAP3 in regulating cell death, highlighting its potential as a therapeutic target for lung cancer treatment.

Long non-coding RNA H19 promotes proliferation in hepatocellular carcinoma cells via H19/miR-107/CDK6 axis.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide; nevertheless, current therapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms in HCC biology could yield important insights for the intervention of novel therapies. Recently, various studies have reported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19; however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disrupted HCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19 yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulation of CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the 3' untranslated region (3' UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), and the dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107 activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves as a molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this study demonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppression as a novel approach for HCC treatment.

Pharmacological Ascorbate Elicits Anti-Cancer Activities against Non-Small Cell Lung Cancer through Hydrogen-Peroxide-Induced-DNA-Damage.

Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH-; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH- on NSCLC and elucidate its underlying mechanisms. P-AscH- treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH- in NSCLC therapy. Treatments with P-AscH- increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH- on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH-. The data from this current research advance our understanding of P-AscH- in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.

Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiellapneumoniae infections.

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

CAMSAP3 negatively regulates lung cancer cell invasion and angiogenesis through nucleolin/HIF-1α mRNA complex stabilization.

AIMS: Cancer metastasis is a major cause of lung cancer-related mortality, so identification of related molecular mechanisms is of interest. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) has been implicated in lung cancer malignancies; however, its role in metastatic processes, including invasion and angiogenesis, is largely unknown. MAIN METHOD: The clinical relevance of CAMSAP3 expression in lung cancer was evaluated. The relevance of CAMSAP3 expression to in vitro cell invasion and angiogenesis was assessed in human lung cancer cells and endothelial cells, respectively. The molecular mechanism was identified by qRT-PCR, immunoprecipitation, mass spectrometry, and RNA immunoprecipitation. The in vivo metastatic and angiogenic activities of lung cancer cells were assessed. KEY FINDINGS: Low CAMSAP3 expression was found in malignant lung tissues and strongly correlated with a poor prognosis in lung adenocarcinoma (LUAD). CAMSAP3-knockout NSCLC exhibited high invasive ability, and CAMSAP3 knockout induced HUVEC proliferation and tube formation; these effects were significantly attenuated by reintroduction of exogenous wild-type CAMSAP3. Mechanistically, in the absence of CAMSAP3, the expression of hypoxia-inducible factor-1α (HIF-1α) was upregulated, which increased the levels of downstream HIF-1α targets such as vascular endothelial growth factor A (VEGFA) and matrix metalloproteinases (MMPs) 2 and 9. Proteomic analysis revealed that nucleolin (NCL) bound to CAMSAP3 to regulate HIF-1α mRNA stabilization. In addition, CAMSAP3-knockout lung cancer cells displayed highly aggressive behavior in metastasis and angiogenesis in vivo. SIGNIFICANCE: This study reveals that CAMSAP3 plays a negative regulatory role in lung cancer cell metastatic behavior both in vitro and in vivo through NCL/HIF-1α mRNA complex stabilization.

mTORC2 interactome and localization determine aggressiveness of high-grade glioma cells through association with gelsolin.

mTOR complex 2 (mTORC2) has been implicated as a key regulator of glioblastoma cell migration. However, the roles of mTORC2 in the migrational control process have not been entirely elucidated. Here, we elaborate that active mTORC2 is crucial for GBM cell motility. Inhibition of mTORC2 impaired cell movement and negatively affected microfilament and microtubule functions. We also aimed to characterize important players involved in the regulation of cell migration and other mTORC2-mediated cellular processes in GBM cells. Therefore, we quantitatively characterized the alteration of the mTORC2 interactome under selective conditions using affinity purification-mass spectrometry in glioblastoma. We demonstrated that changes in cell migration ability specifically altered mTORC2-associated proteins. GSN was identified as one of the most dynamic proteins. The mTORC2-GSN linkage was mostly highlighted in high-grade glioma cells, connecting functional mTORC2 to multiple proteins responsible for directional cell movement in GBM. Loss of GSN disconnected mTORC2 from numerous cytoskeletal proteins and affected the membrane localization of mTORC2. In addition, we reported 86 stable mTORC2-interacting proteins involved in diverse molecular functions, predominantly cytoskeletal remodeling, in GBM. Our findings might help expand future opportunities for predicting the highly migratory phenotype of brain cancers in clinical investigations.

Inhibition of histone deacetylase 6 destabilizes ERK phosphorylation and suppresses cancer proliferation via modulation of the tubulin acetylation-GRP78 interaction.

BACKGROUND: The leading cause of cancer-related mortality worldwide is lung cancer, and its clinical outcome and prognosis are still unsatisfactory. The understanding of potential molecular targets is necessary for clinical implications in precision diagnostic and/or therapeutic purposes. Histone deacetylase 6 (HDAC6), a major deacetylase enzyme, is a promising target for cancer therapy; however, the molecular mechanism regulating cancer pathogenesis is largely unknown. METHODS: The clinical relevance of HDAC6 expression levels and their correlation with the overall survival rate were analyzed based on the TCGA and GEO databases. HDAC6 expression in clinical samples obtained from lung cancer tissues and patient-derived primary lung cancer cells was evaluated using qRT-PCR and Western blot analysis. The potential regulatory mechanism of HDAC6 was identified by proteomic analysis and validated by immunoblotting, immunofluorescence, microtubule sedimentation, and immunoprecipitation-mass spectrometry (IP-MS) assays using a specific inhibitor of HDAC6, trichostatin A (TSA) and RNA interference to HDAC6 (siHDAC6). Lung cancer cell growth was assessed by an in vitro 2-dimensional (2D) cell proliferation assay and 3D tumor spheroid formation using patient-derived lung cancer cells. RESULTS: HDAC6 was upregulated in lung cancer specimens and significantly correlated with poor prognosis. Inhibition of HDAC6 by TSA and siHDAC6 caused downregulation of phosphorylated extracellular signal-regulated kinase (p-ERK), which was dependent on the tubulin acetylation status. Tubulin acetylation induced by TSA and siHDAC6 mediated the dissociation of p-ERK on microtubules, causing p-ERK destabilization. The proteomic analysis demonstrated that the molecular chaperone glucose-regulated protein 78 (GRP78) was an important scaffolder required for p-ERK localization on microtubules, and this phenomenon was significantly inhibited by either TSA, siHDAC6, or siGRP78. In addition, suppression of HDAC6 strongly attenuated an in vitro 2D lung cancer cell growth and an in vitro 3D patient derived-lung cancer spheroid growth. CONCLUSIONS: HDAC6 inhibition led to upregulate tubulin acetylation, causing GRP78-p-ERK dissociation from microtubules. As a result, p-ERK levels were decreased, and lung cancer cell growth was subsequently suppressed. This study reveals the intriguing role and molecular mechanism of HDAC6 as a tumor promoter, and its inhibition represents a promising approach for anticancer therapy.

Helicene-Hydrazide Encapsulated Ethyl Cellulose as a Potential Fluorescence Sensor for Highly Specific Detection of Nonanal in Aqueous Solutions and a Proof-of-Concept Clinical Study in Lung Fluid.

Over the past years, lung cancer has been one of the vital cancer-related mortalities worldwide and has inevitably exhibited the highest death rate with the subsequent need for facile and convenient diagnosis approaches to identify the severity of cancer. Previous research has reported long-chain aldehyde compounds such as hexanal, heptanal, octanal, and nonanal as potential biomarkers of lung cancer. Herein, the helicene dye-encapsulated ethyl cellulose (EC@dye-NH) nanosensors have been applied for the potentially sensitive and specific detection of long-chain aldehydes in aqueous media. The sensors contain the intrinsic hydrazide group of dye-NH, which is capable of reacting an aldehyde group via imine formation and the EC backbone. This offers the synergistic forces of hydrophobic interactions with alkyl long-chain aldehydes, which could induce self-assembly encapsulation of EC@dye-NH nanosensors and strong fluorescence responses. The addition of long-chain aldehyde would induce the complete micellar-like nanoparticle formation within 15 min in acetate buffer pH 5.0. The limit of detection (LOD) values of EC@dye-NH nanosensors toward heptanal, octanal, and nonanal were 40, 100, and 10 µM, respectively, without interference from the lung fluid matrices and short-chain aldehydes. For practical applicability, this sensing platform was developed for quantification of the long-chain aldehydes in lung fluid samples with 98-101% recoveries. This EC@dye-NH nanosensor was applied to quantify nonanal contents in lung fluid samples. The results of this method based on EC@dye-NH nanosensors were then validated using standard gas chromatography-mass spectrometry (GC-MS), which gave results consistent with the proposed method. With intracellular imaging application, the EC@dye-NH nanosensors demonstrated excellent intracellular uptake and strong green fluorescence emission upon introducing the nonanal into the lung cancer cells (A549). Thus, the developed nanosensing approach served as the potential fluorescent probes in medical and biological fields, especially for lung cancer disease diagnosis based on highly selective and sensitive detection of long-chain aldehydes.

Phosphoproteomic Analysis Defines BABAM1 as mTORC2 Downstream Effector Promoting DNA Damage Response in Glioblastoma Cells.

Glioblastoma (GBM) is a devastating primary brain cancer with a poor prognosis. GBM is associated with an abnormal mechanistic target of rapamycin (mTOR) signaling pathway, consisting of two distinct kinase complexes: mTORC1 and mTORC2. The complexes play critical roles in cell proliferation, survival, migration, metabolism, and DNA damage response. This study investigated the aberrant mTORC2 signaling pathway in GBM cells by performing quantitative phosphoproteomic analysis of U87MG cells under different drug treatment conditions. Interestingly, a functional analysis of phosphoproteome revealed that mTORC2 inhibition might be involved in double-strand break (DSB) repair. We further characterized the relationship between mTORC2 and BRISC and BRCA1-A complex member 1 (BABAM1). We demonstrated that pBABAM1 at Ser29 is regulated by mTORC2 to initiate DNA damage response, contributing to DNA repair and cancer cell survival. Accordingly, the inactivation of mTORC2 significantly ablated pBABAM1 (Ser29), reduced DNA repair activities in the nucleus, and promoted apoptosis of the cancer cells. Furthermore, we also recognized that histone H2AX phosphorylation at Ser139 (γH2AX) could be controlled by mTORC2 to repair the DNA. These results provided a better understanding of the mTORC2 function in oncogenic DNA damage response and might lead to specific mTORC2 treatments for brain cancer patients in the future.

In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2.

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.

IRF7-deficient MDCK cell based on CRISPR/Cas9 technology for enhancing influenza virus replication and improving vaccine production.

The influenza virus is a cause of seasonal epidemic disease and enormous economic injury. The best way to control influenza outbreaks is through vaccination. The Madin-Darby canine kidney cell line (MDCK) is currently approved to manufacture influenza vaccines. However, the viral load from cell-based production is limited by host interferons (IFN). Interferon regulating factor 7 (IRF7) is a transcription factor for type-I IFN that plays an important role in regulating the anti-viral mechanism and eliminating viruses. We developed IRF7 knock-out MDCK cells (IRF7-/ - MDCK) using CRISPR/Cas9 technology. The RNA expression levels of IRF7 in the IRF7-/ - MDCK cells were reduced by 94.76% and 95.22% under the uninfected and infected conditions, respectively. Furthermore, the IRF7 protein level was also significantly lower in IRF7-/ - MDCK cells for both uninfected (54.85% reduction) and viral infected conditions (32.27% reduction) compared to WT MDCK. The differential expression analysis of IFN-related genes demonstrated that the IRF7-/ - MDCK cell had a lower interferon response than wildtype MDCK under the influenza-infected condition. Gene ontology revealed down-regulation of the defense response against virus and IFN-gamma production in IRF7-/ - MDCK. The evaluation of influenza viral titers by RT-qPCR and hemagglutination assay (HA) revealed IRF7-/ - MDCK cells had higher viral titers in cell supernatant, including A/pH1N1 (4 to 5-fold) and B/Yamagata (2-fold). Therefore, the IRF7-/ - MDCK cells could be applied to cell-based influenza vaccine production with higher capacity and efficiency.

MicroRNA-223 Suppresses Human Hepatic Stellate Cell Activation Partly via Regulating the Actin Cytoskeleton and Alleviates Fibrosis in Organoid Models of Liver Injury.

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate target mRNA expression, and altered expression of miRNAs is associated with liver pathological conditions. Recent studies in animal models have shown neutrophil/myeloid-specific microRNA-223 (miR-223) as a key regulator in the development of various liver diseases including fibrosis, where hepatic stellate cells (HSCs) are the key player in pathogenesis. However, the precise roles of miR-223 in human HSCs and its therapeutic potential to control fibrosis remain largely unexplored. Using primary human HSCs, we demonstrated that miR-223 suppressed the fibrogenic program and cellular proliferation while promoting features of quiescent HSCs including lipid re-accumulation and retinol storage. Furthermore, induction of miR-223 in HSCs decreased cellular motility and contraction. Mechanistically, miR-223 negatively regulated expression of smooth muscle α-actin (α-SMA) and thus reduced cytoskeletal activity, which is known to promote amplification of fibrogenic signals. Restoration of α-SMA in miR-223-overexpressing HSCs alleviated the antifibrotic effects of miR-223. Finally, to explore the therapeutic potential of miR-233 in liver fibrosis, we generated co-cultured organoids of HSCs with Huh7 hepatoma cells and challenged them with acetaminophen (APAP) or palmitic acid (PA) to induce hepatotoxicity. We showed that ectopic expression of miR-223 in HSCs attenuated fibrogenesis in the two human organoid models of liver injury, suggesting its potential application in antifibrotic therapy.

High prevalence of mgrB-mediated colistin resistance among carbapenem-resistant Klebsiella pneumoniae is associated with biofilm formation, and can be overcome by colistin-EDTA combination therapy.

The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.

COVID-19 active case findings based on self-collected saliva samples with CRISPR-Cas12a detection.

COVID-19 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus affecting the world population. Early detection has become one of the most successful strategies to alleviate the epidemic and pandemic of this contagious coronavirus. Surveillance testing programs have been initiated in many countries worldwide to prevent the outbreak of COVID-19. In this study, we demonstrated that our previously established clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a-based assay could detect variants of concern during 2021 in Thailand, including Alpha, Beta, and Delta strains as well as Omicron strain in early 2022. In combination with the newly designed saliva collection funnel, we established a safe, simple, economical, and efficient self-collection protocol for the COVID-19 screening process. We successfully utilized the assay in an active case finding with a total number of 578 asymptomatic participants to detect the SARS-CoV-2 in saliva samples. We finally demonstrated that the validation and evaluation in a large-scale setting could provide valuable information and elaborate the practicality of the test in real-world settings. Our optimized protocol yielded effective results with high sensitivity, specificity, and diagnostic accuracy (96.86%). In addition, this study demonstrates COVID-19 active case findings in low-resource settings, which would be feasible and attractive for surveillance and outbreak prevention in the future.

cGAS deficiency enhances inflammasome activation in macrophages and inflammatory pathology in pristane-induced lupus.

Introduction: Type I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms. Methods: This study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis. Results: The Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages. Conclusion: These findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.

Multidrug-resistant Neisseria gonorrhoeae infection in heterosexual men with reduced susceptibility to ceftriaxone, first report in Thailand.

The global rapid emergence of azithromycin/ceftriaxone resistant Neisseria gonorrhoeae threatens current recommend azithromycin/ceftriaxone dual therapy for gonorrhea to ensure effective treatment. Here, we identified the first two N. gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Among 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, two isolates (NG-083 and NG-091) from urethral swab in male heterosexual patients had reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L). Both were multidrug resistant and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). NG-083 and NG-091 remained susceptible to azithromycin (MIC of 1 mg/L and 0.5 mg/L, respectively). Reduced susceptibility to ceftriaxone was associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region. NG-083 belonged to sequence type (ST) 7235 and NG-091 has new allele number of tbpB with new ST. Molecular docking revealed ceftriaxone weakly occupied the active site of mosaic XXXIV penicillin-binding protein 2 variant in both isolates. Molecular epidemiology results revealed that both isolates display similarities with isolates from UK, USA, and The Netherlands. These first two genetically related gonococcal isolates with decreased ceftriaxone susceptibility heralds the threat of treatment failure in Thailand, and importance of careful surveillance.

Novel colistin-EDTA combination for successful eradication of colistin-resistant Klebsiella pneumoniae catheter-related biofilm infections.

Development of an effective therapy to overcome colistin resistance in Klebsiella pneumoniae, a common pathogen causing catheter-related biofilm infections in vascular catheters, has become a serious therapeutic challenge that must be addressed urgently. Although colistin and EDTA have successful roles for eradicating biofilms, no in vitro and in vivo studies have investigated their efficacy in catheter-related biofilm infections of colistin-resistant K. pneumoniae. In this study, colistin resistance was significantly reversed in both planktonic and mature biofilms of colistin-resistant K. pneumoniae by a combination of colistin (0.25-1 µg/ml) with EDTA (12 mg/ml). This novel colistin-EDTA combination was also demonstrated to have potent efficacy in eradicating colistin-resistant K. pneumoniae catheter-related biofilm infections, and eliminating the risk of recurrence in vivo. Furthermore, this study revealed significant therapeutic efficacy of colistin-EDTA combination in reducing bacterial load in internal organs, lowering serum creatinine, and protecting treated mice from mortality. Altered in vivo expression of different virulence genes indicate bacterial adaptive responses to survive in hostile environments under different treatments. According to these data discovered in this study, a novel colistin-EDTA combination provides favorable efficacy and safety for successful eradication of colistin-resistant K. pneumonia catheter-related biofilm infections.

CAMSAP3 depletion induces lung cancer cell senescence-associated phenotypes through extracellular signal-regulated kinase inactivation.

BACKGROUND: Cellular senescence is an aging-related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a minus-end microtubule-stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood. METHODS: The function of CAMSAP3 on the regulation of cellular senescence-associated phenotypes in human non-small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence-associated phenotypes were observed by SA-ß-Gal staining. Quantitative RT-PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3-ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed. RESULTS: CAMSAP3 knockout promotes lung cancer cell senescence-associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p-ERK) was markedly downregulated in CAMSAP3-deleted cells, suppressing cyclin D1 expression levels, and full-length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3-modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p-ERK and cyclin D1, resulting in a senescence-like phenotype. CONCLUSION: This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence-associated phenotypes via the modulation of p-ERK/cyclin D1 signaling.

Machine Learning-Driven and Smartphone-Based Fluorescence Detection for CRISPR Diagnostic of SARS-CoV-2.

Rapid, accurate, and low-cost detection of SARS-CoV-2 is crucial to contain the transmission of COVID-19. Here, we present a cost-effective smartphone-based device coupled with machine learning-driven software that evaluates the fluorescence signals of the CRISPR diagnostic of SARS-CoV-2. The device consists of a three-dimensional (3D)-printed housing and low-cost optic components that allow excitation of fluorescent reporters and selective transmission of the fluorescence emission to a smartphone. Custom software equipped with a binary classification model has been developed to quantify the acquired fluorescence images and determine the presence of the virus. Our detection system has a limit of detection (LoD) of 6.25 RNA copies/µL on laboratory samples and produces a test accuracy of 95% and sensitivity of 97% on 96 nasopharyngeal swab samples with transmissible viral loads. Our quantitative fluorescence score shows a strong correlation with the quantitative reverse transcription polymerase chain reaction (RT-qPCR) Ct values, offering valuable information of the viral load and, therefore, presenting an important advantage over nonquantitative readouts.

Comparative performance of CRISPR-Cas12a assays for SARS-CoV-2 detection tested with RNA extracted from clinical specimens.

COVID-19 pandemic caused by SARS-CoV-2 infection continue to cause the morbidity and mortality in many countries. Limitations of the gold standard qRT-PCR for diagnosis of this infection includes need for expensive equipment, specialized molecular laboratory, and experienced staff. Currently, CRISPR-based diagnostic method was approved by the U.S. FDA for rapid detection. Several studies developed SARS-CoV-2 detection based on CRISPR-Cas12a platform; however, the validations with RNA extracted from clinical specimens were limited. Therefore, this study evaluated the clinical performance of previously described CRISPR-Cas12a based diagnostic assays for SARS-CoV-2. According to the results, the CRISPR-Cas12a assays on N1 and S genes provided diagnostic accuracy (≥ 95 %) comparable to the qRT-PCR results. The assays with E, N2 and S genes yielded acceptable sensitivity of detection (≥ 95 %) whereas N1 and S genes provided outstanding specificity of detection (100 %). Preferably, multiple target genes should be detected by using CRISPR-Cas12a to ensure the most effective SARS-CoV-2 detection. Therefore, the N1 and S genes would be attractive target genes for SARS-CoV-2 detection based on CRISPR-Cas12a.