Rapid detection of Mycobacterium tuberculosis based on cyp141 via real-time fluorescence loop-mediated isothermal amplification (cyp141-RealAmp).

Background: The rapid detection of Mycobacterium tuberculosis (MTB) is essential for controlling tuberculosis. Methods We designed a portable thermocycler-based real-time fluorescence loop-mediated isothermal amplification assay (cyp141-RealAmp) using six oligonucleotide primers derived from cyp141 to detect MTB. A combined number of 213 sputum samples (169 obtained from clinically diagnosed cases of pulmonary TB and 44 from a control group without tuberculosis) underwent Acid-fast bacillus (AFB) smear, culture, Xpert MTB/RIF assays, and cyp141-RealAmp assay. Results: By targeting MTB cyp141, this technique could detect as low as 10 copies/reaction within 30 min, and it was successfully rejected by other mycobacteria and other bacterial species tested. Of the 169 patients, there was no statistical difference between the detection rate of cyp141-RealAmp (92.90%, 95% CI: 89.03-96.07) and that of Xpert MTB/RIF (94.67%, 95% CI: 91.28-98.06) (P > 0.05), but both were statistically higher than that of culture (65.68%, 95% CI: 58.52-72.84) (P< 0.05) and AFB (57.40%, 95% CI: 49.94-64.86) (P< 0.05). Both cyp141-RealAmp and Xpert MTB/RIF had a specificity of 100%. Furthermore, a high concordance between cyp141-RealAmp and Xpert MTB/RIF was found (Kappa = 0.89). Conclusion: The cyp141-RealAmp assay was shown to be effective, responsive, and accurate in this study. This method offers a prospective strategy for the speedy and precise detection of MTB.

Fatigue crack-based strain sensors achieving flow detection and motion monitoring for reconnaissance robot applications.

Crack-based flexible strain sensors with ultra-high sensitivity under tiny strain are highly desired for environmental perception and motion detection of novel flexible and miniature robots. However, previously reported methods for fabricating crack patterns have often sacrificed the cyclic stability of the sensor, leading to a trade-off relationship between the sensitivity and the cyclic stability. Here, a universal and simple strategy based on fatigue loading with an ultra-large cumulative strain of up to ∼1.2 × 107%, rather than the traditionally quasi-static pre-overloading methods, is proposed to introduce channel cracks in the sensing layer without sacrificing the cyclic stability. The developed flexible strain sensors exhibit high strain-sensitivity (gauge factor = 5798) under tiny strain (< 3%), high cyclic stability (15 000 cycles) and a low strain detecting limit (0.02%). Furthermore, a leaf-like mechanosensor is developed using the fatigue crack-based strain sensor for the realization of multifunctional applications in environment perception and micro-motion detection. Brilliant airflow sensing performance with a wide sensing range (0.93-11.93 m s-1) and a fast response time (0.28 s) for amphibious applications is demonstrated. This work provides a new strategy for overcoming limits of crack-based flexible strain sensors and the developed leaf-like mechanosensor shows great application potential in miniature and flexible reconnaissance robots.

Evaluation of an identification method for the SARS-CoV-2 Delta variant based on the amplification-refractory mutation system.

The Delta variant of SARS-CoV-2 dominated the COVID-19 pandemic due to its high viral replication capacity and immune evasion, causing massive outbreaks of cases, hospitalizations, and deaths. Currently, variant identification is performed mainly by sequencing. However, the high requirements for equipment and operators as well as its high cost have limited its application in underdeveloped regions. To achieve an economical and rapid method of variant identification suitable for undeveloped areas, we applied an amplification-refractory mutation system (ARMS) based on PCR for the detection of novel coronavirus variants. The results showed that this method could be finished in 90 min and detect as few as 500 copies/mL and not react with SARS-Coronavirus, influenza A H1N1(2009), and other cross-pathogens or be influenced by fresh human blood, α- interferon, and other interfering substances. In a set of double-blind trials, tests of 262 samples obtained from patients confirmed with Delta variant infection revealed that our method was able to accurately identify the Delta variant with high sensitivity and specificity. In conclusion, the ARMS-PCR method applied in Delta variant identification is rapid, sensitive, specific, economical, and suitable for undeveloped areas. In our future study, ARMS-PCR will be further applied in the identification of other variants, such as Omicron.

Identification of integrase inhibitor-related drug resistance mutations in newly diagnosed ART-naïve HIV patients.

BACKGROUND: In China, the recommended treatment regimens for HIV-infected individuals were tenofovir in combination with lamivudine or emtricitabine as NRTIs, efavirenz or rilpivirine as NNRTIs, lopinavir/ritonavir as protease inhibitors, and raltegravir or dolutegravir as INSTIs. The development of drug resistance increases the risk of viral rebound, opportunistic infections, and ultimately treatment failure such that the early detection of resistance is ideal. This study was developed to explore primary drug resistance characteristics and genotypic distributions in newly diagnosed antiretroviral therapy (ART)-naïve HIV-1 patients in Nanjing with the goal of establishing a basis for their individualized treatment in the clinic. METHODS: Samples of serum were collected from newly diagnosed ART-naïve HIV patients from the Second Hospital of Nanjing between May 2021 and May 2022. The HIV-1 integrase (IN), protease (PR), and reverse transcriptase (RT) gene coding sequences were amplified from these samples, sequenced, and assessed for drug resistance-related mutations. RESULTS: Major integrase resistance-related mutations were detected in 4/360 amplified samples, with 5 other patient samples exhibiting accessory resistance mutations. The overall prevalence of PR and RT inhibitor-related transmitted drug resistance mutations (TDRMs) in this patient population was 16.99% (61/359). The most common mutations were non-nucleoside reverse transcriptase inhibitor-related mutations (51/359; 14.21%), followed by those associated with nucleoside reverse transcriptase inhibitors (7/359; 1.95%) and protease inhibitors (7/359; 1.95%). Dual-resistant strains were also observed in a subset of patients. CONCLUSIONS: In summary, this study is the first to have surveyed the prevalence of integrase inhibitor resistance-related mutations and other drug resistance-related mutations among newly diagnosed ART-naïve HIV-positive patients in Nanjing, China. These results highlight the need for further molecular surveillance-based monitoring of the HIV epidemic in Nanjing.

Epidemiology of Nontuberculous Mycobacteria in Nanjing and MAB_0540 Mutations Associated with Clofazimine Resistance in Mycobacterium abscessus.

Background: Nontuberculous mycobacteria (NTM) are easily misdiagnosed as multidrug-resistant tuberculosis (MDR-TB), and treatment drugs are very limited. The main objective of our study was to evaluate the minimal inhibitory concentration (MIC) in vitro of bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), delamanid (DLM), and pretomanid (PA-824) for treatment of M. abscessus and M. intracellulare. Furthermore, we determined whether MAB_1448, MAB_4384, MAB_2299c, MAB_1483, MAB_0540, rplD, rplC, and rrl were related to drug resistance to provide an experimental basis for the use of these five drugs in the treatment of NTM. Methods: We identified sample characteristics of epidemics in 550 patients with suspected NTM infection in Nanjing from 2019 to 2021 using the PCR-reverse spot hybrid method. Furthermore, we evaluated the MIC of BDQ, CFZ, DLM, LZD, and PA-824 against 155 clinical isolates of NTM using the microbroth dilution method. The resistant isolates were sequenced using Sanger sequencing. Results: The top three dominant species of NTM distributed in Nanjing were M. intracellulare, M. avium, and M. abscessus. Notably, the proportion of M. abscessus infections increased. The proportion of M. abscessus increased from 12% in 2019 to 18% in 2021. Demographic analysis showed that female infection rates were substantialy greater than male for M. abscessus (P=0.017, <0.05). Our results demonstrate that NTM are highly sensitive to bedaquiline and clofazimine in vitro. However, delamanid and pretomanid had little effect on M. abscessus and M. intracellulare. In addition, we found 30-41 nucleotide deletion mutations and some novel point mutations in the MAB_0540 gene of M. abscessus that are resistant to clofazimine. Conclusion: Bedaquiline, clofazimine, and linezolid were more successful in vitro treatments against M. abscessus and M. intracellulare. The MAB_0540 mutation may be associated with resistance of M. abscessus to clofazimine.

Numerical Simulation in the Melt Pool Evolution of Laser Powder Bed Fusion Process for Ti6Al4V.

In order to track the free interface of the melt pool and understand the evolution of the melt pool, the flow of fluid, and the interface behavior of gas and liquid, a physical model is developed by using the VOF method in this paper. Its characteristics are a combined heat source model, including a parabolic rotation and a cylindrical distribution, and a powder bed stochastic distributed model with powder particle size. The unit interface between the metallic and gas phase in the laser-powder interaction zone can only be loaded by the heat source. Only the first and second laser scanning tracks are simulated to reduce the calculation time. The simulation results show that process parameters such as laser power and scanning speed have significant effects on the fluid flow and surface morphology in the melt pool, which are in good agreement with the experimental results. Compared with the first track, the second track has larger melt pool geometry, higher melt temperature, and faster fluid flow. The melt flows intensely at the initial position due to the high flow rate in the limited melt space. Because there is enough space for the metal flow, the second track can obtain smooth surface morphology more easily compared to the first track. The melt pool temperature at the laser beam center fluctuates during the laser scanning process. This depends on the effects of the interaction between heat conduction or heat accumulation or the interaction between heat accumulation and violent fluid flow. The temperature distribution and fluid flow in the melt pool benefit the analysis and understanding of the evolution mechanism of the melt pool geometry and surface topography and further allow regulation of the L-PBF process of Ti6Al4V.

SARS-CoV-2 detection using quantum dot fluorescence immunochromatography combined with isothermal amplification and CRISPR/Cas13a.

The development of reliable, sensitive, and fast devices for the diagnosis of COVID-19 is of great importance in the pandemic of the new coronavirus. Here, we proposed a new principle of analysis based on a combination of reverse transcription and isothermal amplification of a fragment of the gene encoding the S protein of the SARS-CoV-2 and the CRISPR/Cas13a reaction for cleavage of the specific probe. As a result, the destroyed probe cannot be detected on an immunochromatographic strip using quantum fluorescent dots. Besides, the results can be obtained by an available and inexpensive portable device. By detecting SARS-CoV-2 negative (n = 25) and positive (n = 62) clinical samples including throat swabs, sputum and anal swabs, the assay showed good sensitivity and specificity of the method and could be completed within 1 h without complicated operation and expensive equipment. These superiorities showed its potential for fast point-of-care screening of SARS-CoV-2 during the outbreak, especially in remote and underdeveloped areas with limited equipment and resources.

Development of a Novel Bioluminescence Pyrophosphate Assay for the High-Sensitivity Detection of Hepatitis B Virus.

The transmission of bloodborne viruses through transfusion remains a major blood supply-related safety concern, with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) being the most important pathogens in this context. Real-time bioluminescent pyrophosphate testing has been developed as a means of readily detecting bacterial cells within particular sample types without requiring the use of expensive or complex instrumentation. The sensitivity of this approach, however, is often limited such that it is not compatible with many potential applications. In this study, we sought to overcome the limitations of this pyrophosphate bioluminescent assay format by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) in place of dATP for PCR amplification, thereby dramatically reducing background signal levels. We leveraged this combination PCR and bioluminescent pyrophosphate assay approach to facilitate HBV detection. This assay yielded a limit of detection of 500 copies/mL, making it more sensitive than traditional bioluminescent assays, about 1000 times more sensitive than that of PCR product analysis by agarose gel electrophoresis, and roughly as sensitive as qPCR as a means of detecting viral DNA. We then used this assay to analyze 100 serum samples, with qPCR being used for result validation. The assay required 100 min to complete, and was able to detect as few as 500 copies/mL of viral DNA. Overall, our approach was rapid, sensitive, and simple, enabling users to readily detect HBV in a reliable and efficient manner.

Amplification-free smartphone-based attomolar HBV detection.

Classical gold standard HBV detection relies on expensive devices and complicated procedures, thus is always restricted in large-scale hospitals and centers for disease control and prevention. To extend HBV detection to primary clinics especially in underdeveloped areas, we design amplification-free smartphone-based attomolar HBV detecting technique based on single molecule sensing. Verified by synthesized HBV target DNA, this technique reaches a detection limit at attomolar concentration (100 aM); and verified by 110 clinical samples, it also reaches a rather high sensitivity of 104 copy/mL (≈2000 IU/mL) with a high accuracy of 93.64% certificated by gold standard HBV detecting devices. Besides, this technique can quantify HBV viral load in 70 min only using portable and inexpensive devices as well as simple operations. Because of its cost-effective, field-portable and operable design, highly sensitive and selective detecting capability and wireless data connectivity, this technique can be potentially used in mobile HBV diagnoses and share HBV epidemic information especially in resource limited situations.

Clinical application of thioredoxin reductase as a novel biomarker in liver cancer.

Hepatic cancer is often amenable to surgery, including percutaneous ablation, trans-arterial chemoembolization. However, in metastatic cases, surgery is often not an effective option. Chemotherapy as a conventional clinical method for treatment of malignant diseases may be useful in such cases, but it is likewise not always able to slow or halt progression, therefore novel approaches for treatment of hepatic cancer are needed. Current research suggests that molecular tumor markers (TM) can play a crucial role for diagnosis and prognostic evaluation of malignancies, and TM such as AFP, CEA, CA19-9 have been reported in many malignant diseases. Thioredoxin reductase (TrxR), a type of anti-oxidant biomarker, has become a TM of significant interest. However, little is known about the above TM and TrxR activity in liver cancer. Therefore, this paper aimed to assess these TM with regards to diagnosis and and monitoring treatment efficacy in both primary and metastatic liver cancer. Our results showed TrxR had superior performance for discriminating between liver cancer patients and healthy controls than AFP, CEA, and CA19-9. TrxR also exhibited superior performance for assessing benefits of chemotherapy regardless if patients had PLC or MLC. Meanwhile, due to diagnostic efficiency of unresponsive chemotherapy patients, TrxR also showed a higher activity levels than other general markers in liver metastasis patients. Our results suggest that application of TrxR in combination with other tumor markers may maximize the efficiency of diagnosis and assessment of therapeutic efficiency, and provide new insights for the clinical application of TrxR as a candidate biomarker for liver cancer.

Telomerase inhibition decreases esophageal squamous carcinoma cell migration and invasion.

Telomerase has been shown to be associated with a variety of cancer types. To elucidate the role of telomerase in esophageal squamous carcinoma (ESCC), tissue samples from 100 patients with ESCC, and paired paracancerous tissues from 75 of these patients, were collected for use in the present study. Using immunohistochemical analysis, the expression of telomerase reverse transcriptase (hTERT) in the cytoplasm of ESCC cells was revealed to be significantly higher compared with that in paracancerous tissues, and no significant difference was observed between hTERT expression in the nucleus of ESCC and paracancerous tissue cells. Combined analysis revealed that the cytoplasmic hTERT-positive rate of patients with ESCC was significantly associated with pathological grade, N stage and Tumor-Node-Metastasis (TNM) stage; these data support the association between hTERT expression and poor patient prognosis. In vitro experiments demonstrated that hTERT knockdown does not inhibit the proliferation of ESCC Kyse410 or Kyse520 cells, but inhibits their migration and invasion abilities. These findings indicate that hTERT expression is associated with ESCC metastasis. Interestingly, decreased colony-formation ability was observed in Kyse410 cells, but not in Kyse520 cells. Collectively, the results of the present study suggest that hTERT may serve as a potential therapeutic target for ESCC.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

Quantitative protein detection using single molecule imaging enzyme-linked immunosorbent assay (iELISA).

Protein detection is a key step in molecular biology research and is required for pathogen and protein marker testing for disease diagnostics. Here, single molecule imaging enzyme-linked immunosorbent assay (iELISA) is proposed to quantitatively measure the porcine circovirus type 2 (PCV2) Cap protein. The monoclonal antibody against PCV2 Cap protein indirectly immobilized on a polyethylene glycol (PEG) passivated slide by biotin-streptavidin interaction is used to capture the PCV2 Cap protein, and the PCV2 Cap protein can be detected in single molecule level according to the fluorescein isothiocyanate (FITC)-labeled secondary antibody using total internal reflection fluorescence microscopy. The single molecule iELISA measurements can be finished within 1 h skipping the time-consuming sample preparation procedures; moreover, it also exhibits excellent protein selectivity and anti-interference capability. With the proposed single molecule iELISA, linear relation between the fluorescent signals and logarithm of target protein concentrations is obtained with the detection limit of 7 ng/mL. Considering its high accuracy in target protein detection with simple procedures and fast speed, it is believed single molecule iELISA can be potentially adopted in fast trace protein detection.

Quantitative Detection and Real-Time Monitoring of Endogenous mRNA at the Single Live Cell Level Using a Ratiometric Molecular Beacon.

Messenger ribonucleic acid (mRNA) plays an important role in various cellular processes. however, traditional techniques cannot realize mRNA detections in live cells as they rely on mRNA purification or cell fixation. To achieve real-time and quantitative mRNA detections at a single live cell level, a single-strand stem-loop-structured ratiometric molecular beacon (RMB) composed of the phosphorothioate-modified loop domain on the 2'-O-methyl RNA backbone with a reporter dye, quencher, and reference dye is proposed to detect the Hsp27 mRNA as a modeled endogenous mRNA. When the RMB hybridizes with the target, the stem-loop structure opens, causing separation of the reporter dye and the quencher and restores the reporter fluorescent signals; therefore, the Hsp27 mRNA can be quantitatively detected according to the ratio of the reporter fluorescent signal to the reference fluorescent signal. Both the phosphorothioate and 2'-O-methyl RNA modifications obviously reduce the nonspecific opening, and the additional reference dye ensures the detection precision using co-localization analysis. Not only does this remove the false-positive signal caused by the nuclease degradation-generated RMB fragment, but it also corrects variations caused by direct measurement of reporter fluorescence intensities at a single cell level owing to inhomogeneity in probe delivery. The designed RMB could detect the Hsp27 mRNA with high signal-to-noise ratio and sensitivity as well as excellent specificity and antidegradation capability proved in vitro and in live cells. Furthermore, it was successfully adopted in subcellular localization, quantitative copy number measurements, and even real-time monitoring of Hsp27 mRNA in live cells, demonstrating that the proposed RMB can be a potential quantitative endogenous mRNA detection tool, especially at a single live cell level.

On-site cell concentration and viability detections using smartphone based field-portable cell counter.

We designed a smartphone based field-portable cell counter combining the smartphone microscope for bright-field image recording and the smartphone application for automatically cell recognition, counting and analysis. To our best knowledge, it is the first time that a smartphone based cell counter can distinguish and count both live and dead cells simultaneously. Compared to the results obtained by hemocytometer, commercial cell counter and flow cytometer, the proposed device was proved to detect cell concentration and viability accurately within the application range between 105 cells/mL and 107 cells/mL. Though multiple fields of view were measured to increase the sampling amount for error reduction, the whole operations including image recording and processing can still be finished rapidly. Moreover, the proposed device is cost-effective with small size of 170 mm × 113 mm × 168 mm containing a built-in power supply. Considering its advantages as high accuracy, fast speed, low cost, long battery life and compact configuration, it is believed the proposed device is a potential tool applied in on-site cell analysis.

Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cells.

Background: Wnt5a is a nontransforming Wnt family member and identified as an oncogenic role on cell motility of breast cancer and glioblastoma. However, Wnt5a signaling in esophageal squamous cell carcinoma (ESCC) progression remains poorly defined. Materials and methods: Immunohistochemistry assays were used to measure the Wnt5a expression in ESCC sections. We evaluated the role of receptor tyrosine kinase-like orphan receptor (ROR)1/2 and RhoA on the invasion of ESCC cells by using cell invasion assay, immunoprecipitation, immunofluorescence, and Rho activation assay. Results: Wnt5a was highly expressed in invasive ESCC tissues compared with that in noninvasive and nonmalignant tissues. In vitro assay showed that sfrp2 (Wnt5a antagonist) largely blocked the invasion but not the colony formation of KYSE410 and KYSE520 ESCC cells. Anti-ROR1 mAb and ROR2-shRNA markedly inhibited the disheveled-associated activator of morphogenesis 1 (DAAM1) activity, RhoA activity, microfilament formation and the invasion of ESCC cells. Fluorescent phalloidin staining experiment showed ROR1/ROR2, receptors of Wnt5a signaling, and regulated the reassembly of actin filaments in ESCC cells. Further experiments showed that ROR1 was strongly associated with ROR2 in KYSE410 cells. The activation of RhoA, not Rac1 or Rac2, was involved in ROR1/ROR2 signaling pathway. By using DAAM1 shRNA, we found that RhoA was downstream of DAAM1, which could be rescued by the overexpression of wild-type DAAM1. This could be further proved by a RhoA inhibitor CCG-1423 which could inhibit the invasion of ESCC cells but not DAAM1 activity. Conclusions: Wnt5a promotes ESCC cell invasion via ROR1 and ROR2 receptors and DAAM1/RhoA signaling pathway.

On-site quantitative Hg2+ measurements based on selective and sensitive fluorescence biosensor and miniaturized smartphone fluorescence microscope.

Mercury is a bio-accumulative and toxic pollutant causing severe damages to human health and environment. Since Hg2+ is the most stable form of mercury, selective and sensitive Hg2+ detection is required. Though classical approaches can realize accurate Hg2+ detection, the complicated instruments and the time-consuming operations inevitably limit their on-site applications. Here, we design a smart Hg2+ detection approach using the fluorescence biosensor, the smartphone fluorescence microscope and the smartphone application for Hg2+ on-site detection. Based on the thymine-Hg2+-thymine coordination chemistry, a selective and sensitive fluorescence biosensor is designed for capturing Hg2+ in aqueous solution; besides, a miniaturized smartphone fluorescence microscope for fluorescence signal collection and an image processing application for quantitative Hg2+ measurements are constructed. A highly specific detection of Hg2+ with a linear relation between 1 nM and 1 µM with a limit of detection of 1 nM is obtained using the smart Hg2+ detection approach. Considering it can realize selective and sensitive quantitative Hg2+ measurements in high precision with simple operations and cost-effective system, it is believed the proposed smart Hg2+ detection approach owns great potentials in Hg2+ detection for routine uses at home and in the field.

Real-time analysis of quantum dot labeled single porcine epidemic diarrhea virus moving along the microtubules using single particle tracking.

In order to study the infection mechanism of porcine epidemic diarrhea virus (PEDV), which causes porcine epidemic diarrhea, a highly contagious enteric disease, we combined quantum dot labeled method, which could hold intact infectivity of the labeled viruses to the largest extent, with the single particle tracking technique to dynamically and globally visualize the transport behaviors of PEDVs in live Vero cells. Our results were the first time to uncover the dynamic characteristics of PEDVs moving along the microtubules in the host cells. It is found that PEDVs kept restricted motion mode with a relatively stable speed in the cell membrane region; while performed a slow-fast-slow velocity pattern with different motion modes in the cell cytoplasm region and near the microtubule organizing center region. In addition, the return movements of small amount of PEDVs were also observed in the live cells. Collectively, our work is crucial for understanding the movement mechanisms of PEDV in the live cells, and the proposed work also provided important references for further analysis and study on the infection mechanism of PEDVs.

Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of Broccoli.

Broccoli is found to be a good source of glucosinolates, which can be hydrolyzed by endogenous myrosinase to obtain chemopreventive isothiocyanates (ITCs); among them, sulforaphane (SF) is the most important agent. Studies have shown that cooking greatly affects the levels of SF and total ITCs in broccoli. However, the stability of these compounds during cooking has been infrequently examined. In this study, we proved that the half-lives of SF and total ITCs during stir-frying were 7.7 and 5.9 min, respectively, while the myrosinase activity decreased by 80% after stir-frying for 3 min; SF and total ITCs were more stable than myrosinase. Thus, the contents of SF and total ITCs decreased during stir-frying largely because myrosinase was destroyed. Subsequently, it was confirmed that compared to direct stir-frying, hydrolysis of glucosinolates in broccoli for 90 min followed by stir-frying increased the SF and total ITC concentration by 2.8 and 2.6 times, respectively. This method provides large quantities of beneficial ITCs even after cooking.

Baicalin alleviates radiation-induced epithelial-mesenchymal transition of primary type II alveolar epithelial cells via TGF-ß and ERK/GSK3ß signaling pathways.

BACKGROUND: Radiation therapy is commonly used to treat thoracic malignancies. However, it may lead to severe lung pneumonitis and ultimately fibrosis. Irradiation has been reported to increase epithelial-mesenchymal transition (EMT) of type II alveolar epithelial cells (AEC), which play an important role in pulmonary fibrosis. The transforming growth factor-ß (TGF-ß) and ERK/glycogen synthase kinase 3ß (GSK3ß) pathways are critically involved in radiation-induced EMT. In the present study, we investigated whether baicalin was a novel therapeutic candidate for radiation-induced EMT in type II AEC. METHODS: Primary type II AEC were isolated and treated with 60Co γ-rays and a series doses of baicalin (2µM, 10µM and 50µM). The ultrastructure and morphology changes were observed by transmission electron microscopy and optical microscopy, respectively. Protein expression was determined by western blotting analysis. Immunofluorescence staining was performed to detect the nuclear translocation of Snail. RESULTS: After irradiation, type II AEC displayed a mesenchymal-like morphology accompanied by a decrease in E-cadherin expression, an increase in the expression of Vimentin and α-SMA. Nuclear translocation of Snail, the activation of TGF-ß/Smad pathway, and the inactivation of GSK3ß were prominent in radiation-treated cells. Baicalin significantly attenuated the effects of radiation on type II AEC. CONCLUSIONS: Baicalin may a useful radioprotective agent through suppressing the EMT of type II AEC.