DYNLT1 promotes mitochondrial metabolism to fuel breast cancer development by inhibiting ubiquitination degradation of VDAC1.

BACKGROUND: Mitochondrial metabolism has been proposed as an attractive target for breast cancer therapy. The discovery of new mechanisms underlying mitochondrial dysfunction will facilitate the development of new metabolic inhibitors to improve the clinical treatment of breast cancer patients. DYNLT1 (Dynein Light Chain Tctex-Type 1) is a key component of the motor complex that transports cellular cargo along microtubules in the cell, but whether and how DYNLT1 affects mitochondrial metabolism and breast cancer has not been reported. METHODS: The expression levels of DYNLT1 were analyzed in clinical samples and a panel of cell lines. The role of DYNLT1 in breast cancer development was investigated using in vivo mouse models and in vitro cell assays, including CCK-8, plate cloning and transwell assay. The role of DYNLT1 in regulating mitochondrial metabolism in breast cancer development is examined by measuring mitochondrial membrane potential and ATP levels. To investigate the underlying molecular mechanism, many methods, including but not limited to Co-IP and ubiquitination assay were used. RESULTS: First, we found that DYNLT1 was upregulated in breast tumors, especially in ER + and TNBC subtypes. DYNLT1 promotes the proliferation, migration, invasion and mitochondrial metabolism in breast cancer cells in vitro and breast tumor development in vivo. DYNLT1 colocalizes with voltage-dependent anion channel 1 (VDAC1) on mitochondria to regulate key metabolic and energy functions. Mechanistically, DYNLT1 stabilizes the voltage-dependent anion channel 1 (VDAC1) by hindering E3 ligase Parkin-mediated VDAC1 ubiquitination and degradation. CONCLUSION: Our data demonstrate that DYNLT1 promotes mitochondrial metabolism to fuel breast cancer development by inhibiting Parkin-mediated ubiquitination degradation of VDAC1. This study suggests that mitochondrial metabolism can be exploited by targeting the DYNLT1-Parkin-VDAC1 axis to improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC).

[Studies and application method and quality control system for detecting hepatitis A virus in water for fruits and vegetables production].

OBJECTIVE: To explore detecting methods and quality control standard system for hepatitis A virus( HAV) in water for fruits and vegetables production. METHODS: 100 µL of coliphage MS2( 2. 5 × 10~9 pfu / mL) was added into each water sample, and positively charged membrane filter method was used to capture virus. The virus extraction efficiency of each sample can be calculated according to standard curve of coliphage. Then the quality control system and real-time RT-PCR method were established. RESULTS: This research compared the extraction efficiency of HAV and coliphage MS2 which were added into the water samples at the same time. The extraction efficiency of HAV was from 1. 24% to 32. 68%, and coliphage MS2 1. 64% to 14. 24%. The efficiency met the requirements of ISO / TS 15216-2-2013. Meanwhile, the HAV in 30 water samples were detected, and one was positive. The extraction efficiency of coliphage MS2 was from1. 24% to 24. 19%, and the standard deviation was 0. 0612. CONCLUSION: This research establish a quality control system to ensure the quality of test results.

[Risk Assessment and Genotyping of Hepatitis A Virus in Fruit and Vegetable Products].

This study explored risk assessment and genotyping of hepatitis A virus(HAV)in fruit and vegetable products. Two hundred and sixteen samples of fruit and vegetable products were examined by real-time RT-PCR. Six samples tested positive for hepatitis A virus, including frozen strawberries, frozen blueberries, frozen diced potatoes, frozen diced apple and frozen raspberries, accounting for 2.8% of the total samples tested. These six HAV isolates were genotyped by nested RT-PCR amplification, and a single band was detected in isolates from frozen diced apple(210-1999)and frozen blueberries(210-2002).These two isolates belong to the HAV IB subtype, based on analysis of evolution and homology. This study provides HAV risk information for fruit and vegetable enterprises and food safety management departments. Furthermore, it lays a foundation for HAV traceability, and provides technical support to ensure product safety for enterprises at critical control points including planting, harvest, processing and packaging. These results provide reliable data for epidemiological diagnosis.

Establishment of a sensitive and specific hyper-branched rolling circle amplification assay and test strip for TSV.

A specific padlock probe (PLP) and detection probe were designed to target the capsid protein gene of Taura syndrome virus (TSV), and a hyper-branched rolling circle amplification (HRCA) assay and a corresponding strip-based test were produced. The reaction time and temperature were optimized for both. The PLPs were linked with the target sequence via T4 DNA ligase under conditions of 37°C for 30 min, followed by reaction with Bst DNA polymerase Large Fragment at 61°C for 30 min, and then the test strip was made using the detection probe. Both the assay and the strip had similarly high accuracy and specificity, and their sensitivity was close to 10 copies, which is 100 times higher than that of conventional RT-PCR. We tested 89 batches of shrimps for TSV to assess the HRCA assay and test strip; the results indicated that the TSV HRCA assay and the test strip are rapid diagnostic tools for detection in the field, and have potential for early diagnosis of TSV.

A sensitive and specific hyperbranched rolling circle amplification assay and test strip for white spot syndrome virus.

White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/µL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.

Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR.

The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

Mechanism of inactivation of murine norovirus-1 by high pressure processing.

Murine norovirus-1 (MNV) is currently the most suitable surrogate for human norovirus. The mechanism of MNV-1 inactivation by high pressure processing (HPP) was investigated. HPP-treated MNV could not bind to its target receptor and therefore could not initiate infection of mouse RAW cells. The integrity of the capsid was not affect by HPP. Partial motif changes of the viral capsid caused by HPP were accessed by induced sensitivity to proteinase K.