Identification of Tunable, Environmentally Responsive Fluorogenic Dyes by High-Throughput Screening.

Small molecule dyes remain essential biological tools, yet only a handful of environmentally responsive fluorogenic small molecules are available for routine characterization of protein state. Here, we report the development and execution of a high throughput screen to identify compounds that increase in fluorescence in response to binding of lipophilic sites of proteins. This effort yielded two small molecules that potently indicate the presence of a range of common proteins and outperform common dyes in differential scanning fluorimetry experiments. Structure activity relationship studies revealed that these two scaffolds can be tuned both for their quantum yields and emission wavelengths. This work affords a straightforward framework for the discovery of new fluorophores and adds two fluorogenic probes to the toolbox for studying protein state.

Different Contributions of embB and ubiA Mutations to Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates.

Objective: To explore the association between the variant mutations within embB and ubiA, and the degree of ethambutol (EMB) resistance of Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods: A total of 146 M. tuberculosis isolates were used to determine the minimum inhibitory concentrations (MICs) of EMB with a 96-well microplate-based assay. The mutations within embB and ubiA among these isolates were identified with DNA sequencing. Moreover, a multivariate regression model and a computer model were established to assess the effects of mutations on EMB resistance. Results: Our data showed that overall 100 isolates exhibited 28 mutated patterns within the sequenced embB and ubiA. Statistical analysis indicated that embB mutations Met306Val, Met306Ile, Gly406Ala, and Gln497Arg, were strongly associated with EMB resistance. Of these mutations, Met306Val and Gln497Arg were significantly associated with high-level EMB resistance. Almost all multiple mutations occurred in high-level EMB-resistant isolates. Although the mutation within ubiA accompanied with embB mutation presented exclusively in EMB-resistant isolates, four single ubiA mutations (Ala39Glu, Ser173Ala, Trp175Cys, and Val283Leu) leading to protein instability were observed in EMB-susceptible isolates. Conclusion: This study highlighted the complexity of EMB resistance. Some individual mutations and multiple mutations within embB and ubiA contributed to the different levels of EMB resistance.

Comparative study of the risk prediction model of early postoperative frailty in elderly enterostomy patients based on machine learning methods.

Objective: Based on machine learning method, four types of early postoperative frailty risk prediction model of enterostomy patients were constructed to compare the performance of each model and provide the basis for preventing early postoperative frailty of elderly patients with enterostomy. Methods: The prospective convenience sampling method was conducted and 362 early postoperative enterostomy patients were selected in three hospitals from July 2020 to November 2023 in Shanghai, four different prediction models of Support Vector Machine (SVM), Bayes, XG Boost, and Logistic regression were used and compared the test effects of the four models (MCC, F1, AUC, and Brier index) to judge the classification performance of the four models in the data of this study. Results: A total of 21 variables were included in this study, and the predictors mainly covered demographic information, stoma-related information, quality of life, anxiety and depression, and frailty. The validated models on the test set are XGBoost, Logistic regression, SVM prediction model, and Bayes on the MCC and F1 scores; on the AUC, XGBoost, Logistic regression, Bayes, and SVM prediction model; on the Brier scores, Bayes, Logistic regression, and XGBoost. Conclusion: XGBoost based on machine learning method is better than SVM prediction model, Logistic regression model and Bayes in sensitivity and accuracy. Quality of life in the early postoperative period can help guide clinical patients to identify patients at high risk of frailty and reduce the incidence of early postoperative frailty in elderly patients with enterostomy.

The potential of carbonic anhydrase enzymes as a novel target for anti-cancer treatment.

Carbonic anhydrase (CA) is a zinc-dependent metal enzyme that maintains the pH and carbon dioxide (CO2) homeostasis in cells by catalyzing the reversible hydration and dehydration of CO2 and bicarbonate (HCO3-). In mammals, there are 16 isozymes of CA existed, namely CAI to CAXIV, but only 15 isozymes are found in humans except CAXV. Human CAs have highly conserved catalytic domains, all of which are distributed in different tissues and play important physiological roles. Changes in their functions may disrupt the typical distribution of CAs throughout human body and therefore CAs can be used as diagnostic biomarkers for many diseases. Furthermore, the expression of CAs is correlated to the progression of numerous tumors, therapeutic sensitivity and patient prognosis. In this review, we discuss thoroughly the structure of CAs, their functional activities in human physiology, dysregulations and diseases related to CAs, and different types of CA inhibitors that can reverse their dysregulation.

Benzyl isothiocyanate ameliorates cognitive function in mice of chronic temporal lobe epilepsy.

Objective: Temporal lobe epilepsy (TLE) is a prevalent refractory partial epilepsy seen in clinical practice, with most cases originating from the hippocampus and being characterized by impaired learning and memory. Oxidative stress plays a direct role in the development of epilepsy and neurodegeneration while promoting cognitive dysfunction. Previous research indicates that benzyl isothiocyanate (BITC) has antioxidative stress properties and contributes to neuroprotection. In this study, we aimed to investigate the neuroprotective effect of BITC on a lithium-pilocarpine-induced temporal lobe epileptic mice model. Methods: We conducted Intellicage learning tests, Morris water maze, open field test, and step-down-type passive avoidance tests, respectively. In addition, body weight and brain-to-body ratio were calculated. Nissl staining, real-time quantitative PCR detection of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H dehydrogenase quinone 1(NQO1) were performed. Content of malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) were determined. Results: Our results demonstrate that BITC enhances cognitive function and motor ability in mice, as determined by Intellicage learning tests, Morris water maze, open field test, and step-down-type passive avoidance tests, respectively. Epilepsy leads to the loss of neurons in the CA3 region, while BITC treatment plays a positive role in neuroprotection, especially in the cortex. In comparison to the control group, the EP group exhibited decreased transcription levels of HO-1 and NQO1, alongside reduced GSH-Px activity, while MDA content was elevated. Conversely, the BITC treatment group, when compared to the EP group, showed enhanced transcription levels of Nrf2, HO-1, and NQO1, along with increased GSH-Px activity, and a decrease in MDA content. Conclusion: In summary, our study provides evidence that BITC can improve cognitive impairments in pilocarpine-induced epileptic mice, demonstrating significant antioxidant effects and neuroprotective properties. This highlights its potential as a phytochemical for managing the sequelae of epilepsy.

Can neurocognition, brain neurotrophic factor, triglyceride, and total cholesterol predict suicidal ideation in first-episode Han Chinese patients with schizophrenia?

OBJECTIVE: Previous studies have suggested that the suicide rate of patients with schizophrenia is high. This study investigates factors influencing suicidal ideation in first-episode schizophrenia patients, focusing on cognitive function, brain-derived neurotrophic factor (BDNF), triglyceride (TG), and total cholesterol (TC) in patients with first-episode schizophrenia. METHODS: A total of 123 patients with first-episode schizophrenia and 38 healthy controls were included in the study. The patients were divided into suicidal and nonsuicidal ideation groups based on the Beck Scale for Suicidal Ideation, and they were assessed with Positive and Negative Syndrome Scale (PANSS). Cognitive function was assessed using the Chinese version of the MATRICS consensus cognitive battery (MCCB) and the serum BDNF, TG, and TC were detected. The main statistical methods include t-test, χ2 test, multivariate logistic regression analysis, receiver operating characteristic (ROC) curve analysis, and the DeLong test. RESULTS: 26.02% of patients exhibited suicidal ideation. Higher PANSS and TC levels were risk factors, while higher MCCB scores and BDNF levels were protective factors. ROC analysis indicated AUCs of 0.630, 0.724, and 0.762 for serum BDNF, PANSS, and MCCB, respectively, with a combined AUC of 0.870. CONCLUSION: Serum BDNF level, PANSS score, and MCCB score can be used as auxiliary predictors of suicidal ideation in schizophrenic patients. Combining these three indicators can effectively predict suicidal ideation in schizophrenic patients.

Inosine pretreatment of pregnant rats ameliorates maternal inflammation-mediated hypomyelination in pups via microglia polarization switch.

Periventricular leukomalacia (PVL) is a neurological condition observed in premature infants, characterized by hypomyelination and activation of microglia. Maternal inflammation-induced brain injury in offspring significantly contributes to the development of PVL. Currently, there are no clinical pharmaceutical interventions available for pregnant women to prevent maternal inflammation-mediated brain injury in their offspring. Inosine has been shown to modulate the immune response in diverse stressful circumstances, such as injury, ischemia, and inflammation. The aim of this investigation was to examine the potential prophylactic impact of inosine on offspring PVL induced by maternal inflammation. This was accomplished by administering a 1 mg/ml inosine solution (40 ml daily) to pregnant Sprague-Dawley (SD) rats for 16 consecutive days prior to their intraperitoneal injection of lipopolysaccharide (350 µg/kg, once a day, for two days). The results showed that maternal inosine pretreatment significantly reversed the reduction in MBP and CNPase (myelin-related markers), CC-1 and Olig2 (oligodendrocyte-related markers) in their PVL pups (P7), suggesting that inosine administration during pregnancy could improve hypomyelination and enhance the differentiation of oligodendrocyte precursor cells (OPCs) in their PVL pups. Furthermore, the protective mechanism of inosine against PVL is closely associated with the activation and polarization of microglia. This is evidenced by a notable reduction in the quantity of IBA 1-positive microglia, a decrease in the level of CD86 (a marker for M1 microglia), an increase in the level of Arg 1 (a marker for M2 microglia), as well as a decrease in the level of pro-inflammatory factors TNF-α, IL-1ß, and IL-6, and an increase in the level of anti-inflammatory factors IL-4 and IL-10 in the brain of PVL pups following maternal inosine pretreatment. Taken together, inosine pretreatment of pregnant rats can improve hypomyelination in their PVL offspring by triggering the M1/M2 switch of microglia.

The effect of telemedicine on stoma-related complications in adults with enterostomy: A systematic review and meta-analysis.

To assess the effect of telemedicine on stoma-related complications in adults with enterostomy, we conducted a meta-analysis to evaluate the effects of the telemedicine group compared to the usual group. Literature searches were performed in PubMed, Embase, Web of Science, The Cochrane Library, China Biology Medicine (CBM), China National Knowledge Infrastructure (CNKI), WanFang and VIP databases from their inception up to October 2023. Two authors independently screened and extracted data from the included and excluded literature according to predetermined criteria. Data collected were subjected to meta-analysis using Review Manager 5.3 software. The final analysis included a total of 22 articles, encompassing 2237 patients (telemedicine group: 1125 patients, usual group: 1112 patients). The meta-analysis results demonstrated that, compared to the usual group, the telemedicine group significantly reduced the overall occurrence of stoma-related complications, with an odds ratio (OR) of 0.22 (95% CI = 0.15-0.32, p < 0.00001). Furthermore, it resulted in a decrease in stoma complications (OR = 0.27, 95% CI = 0.15-0.47, p < 0.00001) and peristomal complications (OR = 0.25, 95% CI = 0.19-0.34, p < 0.00001). Therefore, the existing evidence suggests that the application of telemedicine can reduce the incidence of stoma and peristomal complications, making it a valuable clinical recommendation.

Covalent Targeting As a Common Mechanism for Inhibiting NLRP3 Inflammasome Assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines, including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remain poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit the inflammasome assembly and activity. From this screen, we identify and profile inflammasome inhibition of 20 new covalent compounds across nine different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks the activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular cross-linking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, further support the continued development of electrophilic compounds that target reactive cysteine residues on NLRP3 to regulate its activation and activity.

A highly reversible force-assisted Li - CO2 battery based on piezoelectric effect of Bi0.5Na0.5TiO3 nanorods.

The use of light-assisted cathode is regarded as an effective approach to reduce the overpotential of lithium carbon dioxide (Li - CO2) batteries. However, the inefficient electron-hole separation and the complex discharge-charge reactions hamper the efficiency of CO2 photocatalytic reaction in battery. Herein, a highly reversible force-assisted Li - CO2 battery has been established for the first time by employing a Bi0.5Na0.5TiO3 nanorods piezoelectric cathode. The high-energy electron and holes generated by the piezoelectric cathode with ultrasonic force can effectively enhance the carbon dioxide reduction reaction (CDRR) and carbon dioxide evolution reaction (CDER) kinetics, thereby reducing the overpotentials during the discharge-charge processes. Moreover, the morphology of the discharge product (Li2CO3) can be modified via the dense surface electrons of the piezoelectric cathode, resulting in the promoted decomposition kinetics of Li2CO3 in charging progress. Thus, the force-assisted Li - CO2 battery with the unique piezoelectric cathode can adjust the output and input energy by ultrasonic wave, and provides an ultra-low charging platform of 3.52 V, and exhibits excellent cycle stability (a charging platform of 3.42 V after 100 h cycles). The investigation of the force-assisted process described herein provides significant insights to solve overpotential in the Li - CO2 batteries system.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may, therefore, display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP-driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP-dependent cellular growth.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

Covalent targeting as a common mechanism for inhibiting NLRP3 inflammasome assembly.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines including IL-1ß and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remains poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit inflammasome assembly and activity. From this screen we identify and profile inflammasome inhibition of 20 new covalent compounds across 9 different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular crosslinking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, suggests that NLRP3 serves as a cellular electrophile sensor important for coordinating inflammatory signaling in response to redox stress. Further, our results support the potential for covalent cysteine modification of NLRP3 for regulating inflammasome activation and activity.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Crosstalk between metabolism and stress-responsive signaling is essential to maintaining cellular homeostasis. One way this crosstalk is achieved is through the covalent modification of proteins by endogenous, reactive metabolites that regulate the activity of key stress-responsive transcription factors such as NRF2. Several metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 regulatory protein KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolic pathways to NRF2 activation. We found that succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 transcriptional signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2.

Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth.

miR-21-5p Inhibits Ferroptosis in Hepatocellular Carcinoma Cells by Regulating the AKT/mTOR Signaling Pathway through MELK.

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent cancers, and its incidence rate is increasing worldwide. At present, there is no ideal treatment for HCC. In recent years, molecular-targeted therapy has shown significant therapeutic benefits for patients. Ferroptosis is a modality of regulated cell death, and previous studies have found that inducing ferroptosis in liver cancer cells can inhibit the progression of liver cancer. The aim of this study is to investigate the regulatory mechanism of miR-21-5p in regulating ferroptosis in HCC cells. Methods: CCK-8 was used to measure cell viability, EdU and colony formation were used to measure cell proliferation, and Transwell assays were used to measure cell migration and invasion. RT-qPCR was used to detect the level of miR-21-5p, Western blotting was used to detect the protein expression level, a dual-luciferase reporter gene assay was used to determine the targeting relationship between miR-21-5p and MELK, and coimmunoprecipitation was used to determine the interaction between MELK and AKT. Results: Overexpression of miR-21-5p and MELK facilitated the viability, proliferation, colony formation, invasion, and migration of HCC cells. Downregulation of miR-21-5p suppressed the level of MELK and the progression of HCC. MELK regulated the AKT/mTOR signaling pathway, causing changes in the levels of GPX4, GSH, FTH1, xCT, heme oxygenase 1(HO-1), reactive oxygen species, and Fe2+ to regulate the ferroptosis of hepatoma cells. Erastin, an inducer of ferroptosis, attenuated the repressive influence of miR-21-5p on ferroptosis in HCC cells. Conclusion: In summary, this study demonstrates that miR-21-5p inhibits the ferroptosis of HCC cells by regulating the AKT/mTOR signaling pathway through MELK.

Evaluation of the correlation between sleep quality and work engagement among nurses in Shanghai during the post-epidemic era.

AIM: To examine the status quo and influencing factors of sleep quality and work engagement of nurses participating in COVID-19 during the post-epidemic era and to study the relationship between them. DESIGN: We conducted a cross-sectional survey and correlational and predictive logic to determine the association between sleep quality and work engagement among nurses in Shanghai during the post-epidemic era. METHODS: This design involved 1060 frontline nurses in Shanghai. The Pittsburgh Sleep Quality Index questionnaire and the Utrecht Work Engagement Scale-9 scales were used for data collection. RESULTS: This study found that the sleep quality of frontline nurses was impaired and the nurses with poor sleep accounted for 48.20% during the post-epidemic era. The work engagement of frontline nurses was at the medium level. Factors affecting nurses' sleep quality were the number of nurse night shifts, family support and nurse health. The factors affecting the nurse work engagement were monthly income, profession title, family support and self-health status. There was a positive correlation between nurses' sleep quality and work engagement.

Integrated transcriptomic and metabolomic analyses of DNCB-induced atopic dermatitis in mice.

AIMS: Atopic dermatitis (AD) is a common chronic inflammatory skin disorder that affects up to 20 % of children and 10 % of adults worldwide; however, the exact molecular mechanisms remain largely unknown. MATERIALS AND METHODS: In this study, we used integrated transcriptomic and metabolomic analyses to study the potential mechanisms of 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like skin lesions. KEY FINDINGS: We found that DNCB induced AD-like skin lesions, including phenotypical and histomorphological alterations and transcriptional and metabolic alterations in mice. A total of 3413 differentially expressed metabolites were detected between DNCB-induced AD-like mice and healthy controls, which includes metabolites in taurine and hypotaurine metabolism, phenylalanine metabolism, biosynthesis of unsaturated fatty acids, tryptophan metabolism, arachidonic acid metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism, and glycerophospholipid metabolism pathways. Furthermore, the differentially expressed genes associated (DEGs) with these metabolic pathways were analyzed using RNA sequencing (RNA-seq), and we found that the expression of pyrimidine metabolism-associated genes was significantly increased. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the glycolysis/gluconeogenesis, glucagon signaling pathway and pentose phosphate pathway-associated metabolic genes were dramatically altered. SIGNIFICANCE: Our results explain the possible mechanism of AD at the gene and metabolite levels and provide potential targets for the development of clinical drugs for AD.

rpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis.

Objective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.