Mitochondrial antioxidant elamipretide improves learning and memory impairment induced by chronic sleep deprivation in mice.

BACKGROUND: The inflammation and synaptic dysfunction induced by mitochondrial dysfunction play essential roles in the learning and memory impairment associated with sleep dysfunction. Elamipretide (SS-31), a novel mitochondrion-targeted antioxidant, was proven to improve mitochondrial dysfunction, the inflammatory response, synaptic dysfunction, and cognitive impairment in models of cerebral ischemia, sepsis, and type 2 diabetes. However, the potential for SS-31 to improve the cognitive impairment induced by chronic sleep deprivation (CSD) and its underlying mechanisms is unknown. METHODS: Adult c57BL/6J mice were subjected to CSD for 21 days using an activity wheel accompanied by daily intraperitoneal injection of SS-31 (5 mg/kg). The novel object recognition and Morris water maze test were used to evaluate hippocampus-dependent cognitive function. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were used to determine the effects of CSD and SS-31 on markers of mitochondria, inflammation response, and synaptic function. Enzyme-linked immunosorbent assays were used to examine the levels of proinflammatory cytokines. RESULTS: SS-31 could improve the cognitive impairment induced by CSD. In particular, SS-31 treatment restored the CSD-induced decrease in sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator alpha levels and the increase in levels nuclear factor kappa-B and inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha. Furthermore, SS-31 significantly increased the levels of brain-derived neurotrophic factor, postsynaptic density protein-95, and synaptophysin in CSD mice. CONCLUSION: Taken together, these results suggest that SS-31 could improve CSD-induced mitochondrial biogenesis dysfunction, inflammatory response, synaptic dysfunction, and cognitive impairment by increasing SIRT1 expression levels.

Novel beagle model of gastric local fibrotic target lesions for the evaluation and training of endoscopic techniques.

BACKGROUND: Novel endoscopic techniques used in the treatment of gastric lesions with local submucosal fibrosis need preclinical evaluation and training due to safety limitations. Therefore, the purpose of our study was to establish an animal model of gastric local fibrotic target lesions and assess its feasibility in the evaluation and training of endoscopic techniques. METHODS: In six experimental beagles, a 50% glucose solution was injected into three submucosal areas of the fundus, body, and antrum of the stomach to create gastric local fibrotic target lesions (experimental group). On post-injection day (PID) 7, the injection sites were assessed endoscopically to confirm the presence of submucosal fibrosis formation, and the dental floss clip traction assisted endoscopic submucosal dissection (DFC-ESD) procedure was performed on the gastric local fibrotic target lesions to confirm its feasibility after endoscopic observation. The normal gastric mucosa of six control beagles underwent the same procedure (control group). All the resected specimens were evaluated by histological examination. RESULTS: All 12 beagles survived without postoperative adverse events. On PID 7, 16 ulcer changes were observed at the injection sites (16/18) under the endoscope, and endoscopic ultrasonography confirmed the local submucosal fibrosis formation in all ulcer lesions. The subsequent DFC-ESD was successfully performed on the 32 gastric target lesions, and the mean submucosal dissection time in the ulcer lesions was greater than that in the normal gastric mucosa (15.3 ± 5.6 vs. 6.8 ± 0.8 min; P < 0.001). There was no difference in rates of en bloc resection, severe hemorrhage, or perforation between the two groups. Histological analysis of the ulcer lesions showed the absence of epithelial or muscularis mucosae and extensive submucosal fibrous tissue proliferations compared with normal gastric mucosa. Overall, endoscopists had high satisfaction with the realism and feasibility of the animal model. CONCLUSION: We developed a novel animal model of gastric local fibrotic target lesions to simulate difficult clinical situations, which strongly appeared to be suitable for the preclinical evaluation and learning of advanced endoscopic techniques.

Antitumor activity of miR-188-3p in gastric cancer is achieved by targeting CBL expression and inactivating the AKT/mTOR signaling.

BACKGROUND: Altered miR-188-3p expression has been observed in various human cancers. AIM: To investigate the miR-188-3p expression, its roles, and underlying molecular events in gastric cancer. METHODS: Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression. Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays. The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay. A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival. A nude mouse tumor cell xenograft assay was used to confirm the in vitro data. RESULTS: MiR-188-3p was found to be lower in the plasma of gastric cancer patients, tissues, and cell lines compared to their healthy counterparts. It was associated with overall survival of gastric cancer patients (P < 0.001), tumor differentiation (P < 0.001), lymph node metastasis (P = 0.033), tumor node metastasis stage (I/II vs III/IV, P = 0.024), and American Joint Committee on Cancer stage (I/II vs III/IV, P = 0.03). Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy. CBL was identified as a direct target of miR-188-3p, with its expression antagonizing the effects of miR-188-3p on gastric cancer (GC) cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway. The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer. CONCLUSION: The current data provides ex vivo, in vitro, and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.

Subsequent maternal sleep deprivation aggravates neurobehavioral abnormalities, inflammation, and synaptic function in adult male mice exposed to prenatal inflammation.

Objective: Studies have suggested that prenatal exposure to inflammation increases the risk of neuropsychiatric disorders, including anxiety, depression, and cognitive dysfunction. Because of anatomical and hormonal alterations, pregnant women frequently experience sleep dysfunction, which can enhance the inflammatory response. The aim of this study was to explore the effects of maternal sleep deprivation on prenatal inflammation exposure-induced behavioral phenotypes in offspring and identify the associated mechanisms. Methods: Pregnant mice received an intraperitoneal injection of lipopolysaccharide (LPS) on gestational day 15 and were subsequently subjected to sleep deprivation during gestational days 15-21. Anxiety-like behavior was evaluated by the open field test and the elevated plus maze test. Depression-like behavior was assessed by the tail suspension test and the forced swimming test. Cognitive function was determined using the Morris water maze test. The levels of markers of inflammation and synaptic function were examined employing general molecular biological techniques. Results: The results showed that prenatal exposure to LPS resulted in anxiety- and depression-like symptoms and learning and memory deficits, and these effects were exacerbated by maternal sleep deprivation. Furthermore, maternal sleep deprivation aggravated the prenatal LPS exposure-induced increase in the expression of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and decrease in the levels of postsynaptic density-95 and synaptophysin in the hippocampus. Discussion: Collectively, these results suggested that maternal sleep deprivation exacerbates anxiety, depression, and cognitive impairment induced by prenatal LPS exposure, effects that were associated with an inflammatory response and synaptic dysfunction.

Role of Cyclin D1b in Inducing Macrophages Toward a Tumor-associated Macrophage-like Phenotype in Murine Breast Cancer.

OBJECTIVE: Tumor-associated macrophages (TAMs) of the M2 phenotype are frequently associated with cancer progression. Invasive cancer cells undergoing epithelial-mesenchymal transition (EMT) have a selective advantage as TAM activators. Cyclin D1b is a highly oncogenic splice variant of cyclin D1. We previously reported that cyclin D1b enhances the invasiveness of breast cancer cells by inducing EMT. However, the role of cyclin D1b in inducing macrophage differentiation toward tumor-associated macrophage-like cells remains unknown. This study aimed to explore the relationship between breast cancer cells overexpressing cyclin D1b and TAMs. METHODS: Mouse breast cancer 4T1 cells were transfected with cyclin D1b variant and co-cultured with macrophage cells in a Transwell coculture system. The expression of characteristic cytokines in differentiated macrophages was detected using qRT-PCR, ELISA and zymography assay. Tumor-associated macrophage distribution in a transplanted tumor was detected by immunofluorescence staining. The proliferation and migration ability of breast cancer cells was detected using the cell counting kit-8 (CCK-8) assay, wound healing assay, Transwell invasion assay, and lung metastasis assay. Expression levels of mRNAs were detected by qRT-PCR. Protein expression levels were detected by Western blotting. The integrated analyses of The Cancer Genome Atlas (TCGA) datasets and bioinformatics methods were adopted to discover gene expression, gene coexpression, and overall survival in patients with breast cancer. RESULTS: After co-culture with breast cancer cells overexpressing cyclin D1b, RAW264.7 macrophages were differentiated into an M2 phenotype. Moreover, differentiated M2-like macrophages promoted the proliferation and migration of breast cancer cells in turn. Notably, these macrophages facilitated the migration of breast cancer cells in vivo. Further investigations indicated that differentiated M2-like macrophages induced EMT of breast cancer cells accompanied with upregulation of TGF-ß1 and integrin ß3 expression. CONCLUSION: Breast cancer cells transfected with cyclin D1b can induce the differentiation of macrophages into a tumor-associated macrophage-like phenotype, which promotes tumor metastasis in vitro and in vivo.

Resveratrol ameliorates maternal separation-induced anxiety- and depression-like behaviors and reduces Sirt1-NF-kB signaling-mediated neuroinflammation.

Maternal separation in early life has a detrimental effect on the physiological and biochemical functions of the brains of offspring and can lead to anxiety- and depression-like behaviors later in life. Resveratrol possesses a variety of pharmacological properties, including anti-inflammatory, anxiolytic, and anti-depressive effects. In rodents, resveratrol can attenuate anxiety- and depression-like behaviors induced by chronic unpredictable mild stress, estrogen deficiency, and lipopolysaccharide. However, whether resveratrol administration during adolescence can counteract these behaviors when they result from maternal separation is unknown. In this study, male C57BL/6J mice were separated from their mothers for 4 h per day from postnatal day 2 (PND 2) to PND 21; starting on PND 61, resveratrol was administered intraperitoneally at 40 mg/(kg/day-1) for 4 weeks. At 3 months of age, anxiety and depression-like behaviors were assessed in the male offspring using a series of tasks consisting of an open field test, an elevated plus maze test, a forced swimming test, and a tail suspension test. The hippocampal levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-alpha (TNF-α) were measured by ELISA, while those of sirtuin 1 (Sirt1) and nuclear factor kappa B (NF-κB) p65 were determined by western blotting and PCR. The results showed that maternal separation led to increased anxiety- and depression-like behaviors, enhanced the levels of pro-inflammatory cytokines, and downregulated the Sirt1/NF-κB signaling pathway in the male offspring; however, these effects could be reversed by treatment with resveratrol. Our findings suggested that resveratrol can ameliorate inflammation and anxiety- and depression-like behaviors induced by maternal separation via the activation of the Sirt1/NF-κB pathway.

Effects of gestational inflammation on age-related cognitive decline and hippocampal Gdnf-GFRα1 levels in F1 and F2 generations of CD-1 Mice.

BACKGROUND: It has been reported that age-associated cognitive decline (AACD) accelerated by maternal lipopolysaccharide (LPS) insult during late pregnancy can be transmitted to the second generation in a sex-specificity manner. In turn, recent studies indicated that glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα1) are critical for normal cognitive function. Based on this evidence, we aimed to explore whether Gdnf-GFRα1 expression contributes to cognitive decline in the F1 and F2 generations of mouse dams exposed to lipopolysaccharide (LPS) during late gestation, and to evaluate also the potential interference effect of pro-inflammatory cytokines. METHODS: During gestational days 15-17, pregnant CD-1 mice (8-10 weeks old) received a daily intraperitoneal injection of LPS (50 µg/kg) or saline (control). In utero LPS-exposed F1 generation mice were selectively mated to produce F2 generation mice. In F1 and F2 mice aged 3 and 15 months, the Morris water maze (MWM) was used to evaluated the spatial learning and memory ability, the western blotting and RT-PCR were used for analyses of hippocampal Gdnf and GFRα1 expression, and ELISA was used to analyse IL-1ß, IL-6 and TNF-α levels in serum. RESULTS: Middle-aged F1 offspring from LPS-treated mothers exhibited longer swimming latency and distance during the learning phase, lower percentage swimming time and distance in targe quadrant during memory phase, and lower hippocampal levels of Gdnf and GFRα1 gene products compared to age-matched controls. Similarly, the middle-aged F2 offspring from the Parents-LPS group had longer swimming latency and distance in the learning phase, and lower percentage swimming time and distance in memory phase than the F2-CON group. Moreover, the 3-month-old Parents-LPS and 15-month-old Parents- and Father-LPS groups had lower GDNF and GFRα1 protein and mRNAs levels compared to the age-matched F2-CON group. Furthermore, hippocampal levels of Gdnf and GFRα1 were correlated with impaired cognitive performance in the Morris water maze after controlling for circulating pro-inflammatory cytokine levels. CONCLUSIONS: Our findings indicate that accelerated AACD by maternal LPS exposure can be transmitted across at least two generations through declined Gdnf and GFRα1 expression, mainly via paternal linage.

Gail model and fifth edition of ultrasound BI-RADS help predict axillary lymph node metastasis in breast cancer-A multicenter prospective study.

RATIONALE AND OBJECTIVES: We aim to assess the performance of the Gail model and the fifth edition of ultrasound BI-RADS (Breast Imaging Reporting and Data System) in breast cancer for predicting axillary lymph node metastasis (ALNM). MATERIALS AND METHODS: We prospectively studied 958 female patients with breast cancer between 2018 and 2019 from 35 hospitals in China. Based on B-mode, color Doppler, and elastography, radiologists classified the degree of suspicion based on the fifth edition of BI-RADS. Individual breast cancer risk was assessed with the Gail model. The association between the US BI-RADS category and the Gail model in terms of ALNM was analyzed. RESULTS: We found that US BI-RADS category was significantly and independently associated with ALNM (P < 0.001). The sensitivity, specificity, and accuracy of BI-RADS category 5 for predicting ALNM were 63.6%, 71.6%, and 68.6%, respectively. Combining the Gail model with the BI-RADS category showed a significantly higher sensitivity than using the BI-RADS category alone (67.8% vs. 63.6%, P < 0.001). The diagnostic accuracy of the BI-RADS category combined with the Gail model was better than that of the Gail model alone (area under the curve: 0.71 vs. 0.50, P < 0.001). CONCLUSION: Based on the conventional ultrasound and elastography, the fifth edition of ultrasound BI-RADS category could be used to predict the ALNM of breast cancer. ALNM was likely to occur in patients with BI-RADS category 5. The Gail model could improve the diagnostic sensitivity of the US BI-RADS category for predicting ALNM in breast cancer.

Environmental enrichment improves declined cognition induced by prenatal inflammatory exposure in aged CD-1 mice: Role of NGPF2 and PSD-95.

Introduction: Research suggests that prenatal inflammatory exposure could accelerate age-related cognitive decline that may be resulted from neuroinflammation and synaptic dysfunction during aging. Environmental enrichment (EE) may mitigate the cognitive and synaptic deficits. Neurite growth-promoting factor 2 (NGPF2) and postsynaptic density protein 95 (PSD-95) play critical roles in neuroinflammation and synaptic function, respectively. Methods: We examined whether this adversity and EE exposure can cause alterations in Ngpf2 and Psd-95 expression. In this study, CD-1 mice received intraperitoneal injection of lipopolysaccharide (50 µg/kg) or normal saline from gestational days 15-17. After weaning, half of the male offspring under each treatment were exposed to EE. The Morris water maze was used to assess spatial learning and memory at 3 and 15 months of age, whereas quantitative real-time polymerase chain reaction and Western blotting were used to measure hippocampal mRNA and protein levels of NGPF2 and PSD-95, respectively. Meanwhile, serum levels of IL-6, IL-1ß, and TNF-α were determined by enzyme-linked immunosorbent assay. Results: The results showed that aged mice exhibited poor spatial learning and memory ability, elevated NGPF2 mRNA and protein levels, and decreased PSD-95 mRNA and protein levels relative to their young counterparts during natural aging. Embryonic inflammatory exposure accelerated age-related changes in spatial cognition, and in Ngpf2 and Psd-95 expression. Additionally, the levels of Ngpf2 and Psd-95 products were significantly positively and negatively correlated with cognitive dysfunction, respectively, particularly in prenatal inflammation-exposed aged mice. Changes in serum levels of IL-6, IL-1ß, and TNF-α reflective of systemic inflammation and their correlation with cognitive decline during accelerated aging were similar to those of hippocampal NGPF2. EE exposure could partially restore the accelerated decline in age-related cognitive function and in Psd-95 expression, especially in aged mice. Discussion: Overall, the aggravated cognitive disabilities in aged mice may be related to the alterations in Ngpf2 and Psd-95 expression and in systemic state of inflammation due to prenatal inflammatory exposure, and long-term EE exposure may ameliorate this cognitive impairment by upregulating Psd-95 expression.

Prenatal exposure to inflammation increases anxiety-like behaviors in F1 and F2 generations: possible links to decreased FABP7 in hippocampus.

Anxiety disorder has a high prevalence, and the risk of anxiety increases with age. Prenatal inflammation during key developmental timepoints can result in long-term changes in anxiety phenotype, even over a lifetime and across generations. However, whether maternal inflammation exposure during late gestation has intergenerational transmission effects on age-related anxiety-like behaviors and the possible underlying mechanisms are largely unknown. Fatty acid binding protein 7 (FABP7) is critical in hippocampal neurogenesis and is closely related to neuropsychiatric diseases, including anxiety disorder. The current study investigated the effects of maternal (F0 generation) lipopolysaccharide administration (50 µg/kg, i.p.) during late gestation on anxiety-like behaviors and FABP7 expression in F1 and F2 offspring, as well as the potential sex-specificity of intergenerational effects. Anxiety-like behaviors were evaluated using open field (OF), elevated plus maze, and black-white alley (BWA) tests at 3 and 13 months of age. The protein and messenger RNA levels of FABP7 in the hippocampus were measured using Western blot and real-time quantitative polymerase chain reaction (PCR), respectively. Overall, gestational LPS exposure in the F0 generation increased anxiety levels and decreased FABP7 expression levels in the F1 generation, which carried over to the F2 generation, and the intergenerational effects were mainly transferred via the maternal lineage. Moreover, hippocampal FABP7 expression was significantly correlated with performance in the battery of anxiety tests. The present study suggested that prenatal inflammation could increase age-related anxiety-like behaviors both in F1 and F2 offspring, and these effects possibly link to the FABP7 expression.

ACR TI-RADS classification combined with number of nodules, halo features optimizes diagnosis and prediction of follicular thyroid cancer.

OBJECTIVES: To investigate the application value of The American College of Radiology (ACR) Thyroid Imaging Reporting and Data System (TI-RADS) category combined with other ultrasound features of nodules in distinguishing follicular thyroid carcinoma (FTC) from thyroid follicular adenoma (FTA). METHODS: We collected and retrospectively analyzed clinical and ultrasound data for 118 and 459 patients with FTCs and FTAs, respectively, at our hospital. Next, we used ACR TI-RADS classification combined with other ultrasound features of nodules to distinguish FTC from FTA. Multivariate Logistic regression was used to screen independent risk factors for FTC, which were subsequently used to construct a nomogram for predicting FTC. RESULTS: ACR TI-RADS categories 4 and 5, unilateral multiple nodules, and halo thickness≥2 mm were independent risk factors for FTC. ACR TI-RADS category combined with number of nodules, halo features of the nodule was a significantly better prediction model for FTC diagnosis (AUC = 0.869) than that of ACR TI-RADS classification alone (AUC = 0.756). CONCLUTIONS: Clinicians need to pay attention to the halo of nodules when distinguishing FTA from FTC. Notably, ACR TI-RADS combined with other nodule ultrasound features has superior predictive performance in diagnosis of FTC compared to ACR TI-RADS classification alone, thus can provide an important reference value for preoperative diagnosis of FTC.

Maternal inflammation induces spatial learning and memory impairment in the F1 and F2 generations of mice via sex-specific epigenetic mechanisms.

Mounting evidence indicates that histone modifications are involved in aging-associated cognitive decline (AACD) and can be transmitted to offspring over multiple generations under conditions of stress. Here, we investigated the effects of maternal sub-chronic inflammation caused by lipopolysaccharide (LPS) on AACD and histone modifications in the F1 and F2 generations of experimental mice as well as the potential sex specificity of intergenerational effects. In brief, F0-generation CD-1 dams were exposed to LPS (50 µg/kg) or saline (CON) during late pregnancy. Subsequently, F1 males and females (at 2 months-of-age) from the LPS treatment group were mated with non-littermates from the LPS group or wild-type mice to produce F2 generations of parental- (F2-LPS2), paternal- (F2M-LPS1) and maternal-origin (F2F-LPS1) mice. Then, CON-F1 males and females were mated with wild-type mice to generate F2 generations of paternal- (F2M-CON1) and maternal-origin (F2F-CON1). Next, we evaluated the cognitive ability and levels of hippocampal H4K12ac and H3K9me3 in the F1 and F2 offspring at 3- and 13 months-of-age. Overall, F1 male and female LPS groups presented with elevated corticosterone (P < 0.001, P = 0.036, P = 0.025, 0.012, respectively) and cytokine responses, poorer cognitive performance (all P < 0.05) and H3K9 hypermethylation and H4K12 hypoacetylation in the dorsal hippocampus (all P < 0.05); these issues were carried over to the F2 generation via the parents, predominantly in the paternal lineage. Moreover, the levels of H3K9me3 and H4K12ac were significant correlated with cognitive performance (all P < 0.05), regardless of whether inflammatory insults had been incurred directly or indirectly. These findings indicated that gestational inflammatory insults in the F0 generation accelerated AACD in the F2 generation, along with H3K9 hypermethylation and H4K12 hypoacetylation in the hippocampus, and that these issues were derived from the F1 parents, especially from the F1 fathers.

Methamphetamine induces thoracic aortic aneurysm/dissection through C/EBPß.

AIMS: Thoracic aortic aneurysm/dissection (TAAD) is a life-threatening disease with diverse clinical manifestations. Although the association between methamphetamine (METH) and TAAD is frequently observed, the causal relationship between METH abuse and aortic aneurysm/dissection has not been established. This study was designed to determine if METH causes aortic aneurysm/dissection and delineate the underlying mechanism. METHODS AND RESULTS: A new TAAD model was developed by exposing METH to SD rats pre-treated with lysyl oxidase inhibitor ß-aminopropionitrile (BAPN). Combination of METH and BAPN caused thoracic aortic aneurysm/dissection in 60% of rats. BAPN+METH significantly increased the expression and activities of both matrix metalloproteinase MMP2 and MMP9, consistent with the severe elastin breakage and dissection. Mechanistically, METH increased CCAAT-enhancer binding protein ß (C/EBPß) expression by enhancing mothers against decapentaplegic homolog 3 (Smad3) and extracellular regulated protein kinase (ERK1/2) signaling. METH also promoted C/EBPß binding to MMP2 and MMP9 promoters. Blocking C/EBPß significantly attenuated METH+BAPN-induced TAAD and MMP2/MMP9 expression. Moreover, BAPN+METH promoted aortic medial smooth muscle cell (SMC) apoptosis through C/EBPß-mediated IGFBP5/p53/PUMA signaling pathways. More importantly, the expression of C/EBPß, MMP2/MMP9, and apoptosis-promoting proteins was increased in the aorta of human patients with thoracic aortic dissection, suggesting that the mechanisms identified in animal study could be relevant to human disease. CONCLUSIONS: Our study demonstrated that METH exposure has a casual effect on TAAD. C/EBPß mediates METH-introduced TAAD formation by causing elastin breakage, medial cell loss and degeneration. Therefore, C/EBPß may be a potential factor for TAAD clinical diagnosis or treatment.

Development and evaluation of a meat mitochondrial metagenomic (3MG) method for composition determination of meat from fifteen mammalian and avian species.

BACKGROUND: Bioassessment and biomonitoring of meat products are aimed at identifying and quantifying adulterants and contaminants, such as meat from unexpected sources and microbes. Several methods for determining the biological composition of mixed samples have been used, including metabarcoding, metagenomics and mitochondrial metagenomics. In this study, we aimed to develop a method based on next-generation DNA sequencing to estimate samples that might contain meat from 15 mammalian and avian species that are commonly related to meat bioassessment and biomonitoring. RESULTS: In this project, we found the meat composition from 15 species could not be identified with the metabarcoding approach because of the lack of universal primers or insufficient discrimination power. Consequently, we developed and evaluated a meat mitochondrial metagenomics (3MG) method. The 3MG method has four steps: (1) extraction of sequencing reads from mitochondrial genomes (mitogenomes); (2) assembly of mitogenomes; (3) mapping of mitochondrial reads to the assembled mitogenomes; and (4) biomass estimation based on the number of uniquely mapped reads. The method was implemented in a python script called 3MG. The analysis of simulated datasets showed that the method can determine contaminant composition at a proportion of 2% and the relative error was < 5%. To evaluate the performance of 3MG, we constructed and analysed mixed samples derived from 15 animal species in equal mass. Then, we constructed and analysed mixed samples derived from two animal species (pork and chicken) in different ratios. DNAs were extracted and used in constructing 21 libraries for next-generation sequencing. The analysis of the 15 species mix with the method showed the successful identification of 12 of the 15 (80%) animal species tested. The analysis of the mixed samples of the two species revealed correlation coefficients of 0.98 for pork and 0.98 for chicken between the number of uniquely mapped reads and the mass proportion. CONCLUSION: To the best of our knowledge, this study is the first to demonstrate the potential of the non-targeted 3MG method as a tool for accurately estimating biomass in meat mix samples. The method has potential broad applications in meat product safety.

The Utility of the Fifth Edition of the BI-RADS Ultrasound Lexicon in Category 4 Breast Lesions: A Prospective Multicenter Study in China.

RATIONALE AND OBJECTIVES: The objective of this study was to evaluate the utility of the fifth edition of the Breast Imaging-Reporting and Data System (BI-RADS) in clinical breast radiology by using prospective multicenter real-time analyses of ultrasound (US) images. MATERIALS AND METHODS: We prospectively studied 2049 female patients (age range, 19-86 years; mean age 46.88 years) with BI-RADS category 4 breast masses in 32 tertiary hospitals. All the patients underwent B-mode, color Doppler US, and US elastography examination. US features of the mass and associated features were described and categorized according to the fifth edition of the BI-RADS US lexicon. The pathological results were used as the reference standard. The positive predictive values (PPVs) of subcategories 4a-4c were calculated. RESULTS: A total of 2094 masses were obtained, including 1124 benign masses (54.9%) and 925 malignant masses (45.1%). For BI-RADS US features of mass shape, orientation, margin, posterior features, calcifications, architectural distortion, edema, skin changes, vascularity, and elasticity assessment were significantly different for benign and malignant masses (p< 0.05). Typical signs of malignancy were irregular shape (PPV, 57.2%), spiculated margin (PPV, 83.7%), nonparallel orientation (PPV, 63.9%), and combined pattern of posterior features (PPV, 60.6%). For the changed or newly added US features, the PPVs for intraductal calcifications were 80%, 56.4% for internal vascularity, and 80% for a hard pattern on elastography. The associated features such as architectural distortion (PPV, 89.3%), edema (PPV, 69.2%), and skin changes (PPV, 76.2%) displayed high predictive value for malignancy. The rate of malignant was 7.4% (72/975) in category 4a, 61.4% (283/461) in category 4b, and 93.0% (570/613) in category 4c. The PPV for category 4b was higher than the likelihood ranges specified in BI-RADS and the PPVs for categories 4a and 4c were within the acceptable performance ranges specified in the fifth edition of BI-RADS in our study. CONCLUSION: Not only the US features of the breast mass, but also associated features, including vascularity and elasticity assessment, have become an indispensable part of the fifth edition of BI-RADS US lexicon to distinguish benign and malignant breast lesions. The subdivision of category 4 lesions into categories 4a, 4b, and 4c for US findings is helpful for further assessment of the likelihood of malignancy of breast lesions.

Metformin combined with endoscopic therapy in patients with familial polyposis associated with carcinoma: A case report.

RATIONALE: Familial adenomatous polyposis (FAP) is an autosomal dominant genetic disease, with a very high cancer rate. At present, endoscopic resection of polyps ≥ 1 cm is often chosen for patients with non-cancerous polyps who are unwilling to undergo surgery, and regular review is conducted. Once the polyps are pathologically confirmed to be cancerous, surgical resection of the diseased large intestine is generally recommended, but surgery often leads to a series of complications. So what do you do with cancer patients who don't want surgery? PATIENT CONCERNS: A 19-year-old woman presented with intermittent hematochezia with abdominal pain. A colonoscopy revealed hundreds of intestinal polyps. DIAGNOSES: The patient had a family history of FAP, and there were hundreds of polyps in the intestine. The pathology was adenomatous, and some polyps became cancerous, which met the diagnostic criteria of FAP. INTERVENTIONS: Endoscopic examination was arranged for the patient, the resection of intestinal polyps ≥ 1 cm was given priority, and other polyps were removed as far as possible. After that, metformin 500 mg orally was given twice a day, and endoscopic follow-up was conducted every 6 months. During each endoscopic follow-up, intestinal polyps ≥ 1 cm were preferred to be removed, and other polyps were removed as far as possible. OUTCOMES: The patient's abdominal pain and blood in the stool disappeared after endoscopic treatment. Cancerous polyps were found at the second and third follow-up visits, but the patient always refused surgical treatment. After 4 years of follow-up, polyp load was significantly reduced, abdominal pain and bloody stool symptoms did not appear again, and imaging examination showed no tumor recurrence and metastasis. LESSONS: Endoscopic polyp resection is an important method to treat the clinical symptoms of FAP. Metformin combined with endoscopic therapy is a good alternative for patients with familial polyposis who do not want surgery. When the polyp is cancerous and the polyp is radically resected by the endoscope, if the patient refuses additional surgery, oral metformin combined with endoscopic follow-up can be considered.

Gail Model Improves the Diagnostic Performance of the Fifth Edition of Ultrasound BI-RADS for Predicting Breast Cancer: A Multicenter Prospective Study.

RATIONALE AND OBJECTIVES: The sonographic appearance of benign and malignant breast nodules overlaps to some extent, and we aimed to assess the performance of the Gail model as an adjunctive tool to ultrasound (US) Breast Imaging Reporting and Data System (BI-RADS) for predicting the malignancy of nodules. MATERIALS AND METHODS: From 2018 to 2019, 2607 patients were prospectively enrolled by 35 health care facilities. An individual breast cancer risk was assessed by the Gail model. Based on B-mode US, color Doppler, and elastography, all nodules were evaluated according to the fifth edition of BI-RADS, and these nodules were all confirmed later by pathology. RESULTS: We demonstrated that the Gail model, age, tumor size, tumor shape, growth orientation, margin, contour, acoustic shadowing, microcalcification, presence of duct ectasia, presence of architectural distortion, color Doppler flow, BI-RADS, and elastography score were significantly related to breast cancer (all p < 0.001). The sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and area under the curve (AUC) for combining the Gail model with the BI-RADS category were 95.6%, 91.3%, 85.0%, 97.6%, 92.8%, and 0.98, respectively. Combining the Gail model with the BI-RADS showed better diagnostic efficiency than the BI-RADS and Gail model alone (AUC 0.98 vs 0.80, p < 0.001; AUC 0.98 vs 0.55, p < 0.001) and demonstrated a higher specificity than the BI-RADS (91.3% vs 59.4%, p < 0.001). CONCLUSION: The Gail model could be used to differentiate malignant and benign breast lesions. Combined with the BI-RADS category, the Gail model was adjunctive to US for predicting breast lesions for malignancy. For the diagnosis of malignancy, more attention should be paid to high-risk patients with breast lesions.

Programmed death-ligand 1 expression in the tumour stroma of colorectal liver oligometastases and its association with prognosis after liver resection.

BACKGROUND: The clinical value of programmed death-ligand 1 (PD-L1) expression in colorectal liver oligometastases (CLOs) remains undefined. This study aimed to detect PD-L1 in the microenvironment of CLOs and determine its association with patient prognosis. METHODS: We collected 126 liver-resection specimens from CLO patients who underwent curative liver resection between June 1999 and December 2016. Immunohistochemistry (IHC) was performed to assess PD-L1 expression in paraffin-embedded specimens. Overall survival (OS) and recurrence-free survival (RFS) were analysed using the Kaplan-Meier method and log-rank test. RESULTS: PD-L1 was mainly expressed in the stroma of liver oligometastases. Patients with high PD-L1 expression had a higher proportion of clinical-risk scores (CRSs) of 2-4 (67.7% vs 40.4%; P = 0.004). With a median 58-month follow-up, patients with high PD-L1 expression had a significantly lower 3-year OS rate (65.5% vs 92.7%; P = 0.001) and 3-year RFS rate (34.7% vs 83.8%; P < 0.001) than patients with low PD-L1 expression. Multivariate Cox analysis demonstrated that high PD-L1 expression (hazard ratio [HR] = 3.581; 95% confidence interval [CI] 2.301-9.972; P = 0.015), CRS 2-4 (HR = 6.960; 95% CI 1.135-42.689; P = 0.036) and increased preoperative CA19-9 (HR = 2.843; 95% CI 1.229-6.576; P = 0.015) were independent risk factors for OS. High PD-L1 expression (HR = 4.815; 95% CI 2.139-10.837; P < 0.001) and lymph-node metastasis (HR = 2.115; 95% CI 1.041-4.297; P = 0.038) were independent risk factors for RFS. CONCLUSION: This study found that PD-L1 was commonly expressed in the tumour stroma of CLOs and high PD-L1 expression was associated with poor prognosis.

Assessment of defecation function after sphincter-saving resection for mid to low rectal cancer: A cross-sectional study.

PURPOSE: Patients with mid-to low-rectal cancer can have various dysfunctions of defecation after sphincter-saving resection. Defecation dysfunction can manifest as incontinence, urgency, or frequent bowel movements, and is called low anterior resection syndrome (LARS). This study aimed to examine LARS score and objective anorectal function indices in Chinese patients receiving sphincter-saving surgery for mid-to low-rectal cancer. METHOD: This was a single-center cross-sectional study of patients undergoing sphincter-saving resection for low- or mid-rectal cancer and had restoration of trans-anal defecation for at least 1 month seen between January 2019 and June 2020. Patients completed a questionnaire regarding clinical characteristics, and Low Anterior Resection Syndrome (LARS) score and high-resolution anorectal manometry (HR-ARM) were used to assess defecation function. Multivariable analysis was used to identify variables significantly associated with defecation dysfunction. RESULTS: 146 patients completed and returned the questionnaires. 25 healthy adults also participated as control group for the anorectal manometry. Approximately 76% of patients developed LARS after surgery, of which 35.6% had major LARS. In these patients, anorectal manometry indices including initial rectal sensory capacity and rectal fecal sensory capacity, were significantly lower than normal. Logistic regression analysis showed that preoperative chemo-radiotherapy and the tumor inferior margins being close to the dentate line, especially 2-5 cm, were independent risk factors for defecation dysfunction after surgery. CONCLUSIONS: Defecation dysfunction is a frequent occurrence after sphincter-saving resection for mid- and low-rectal cancer. Preoperative chemo-radiotherapy and a shorter tumor inferior margins distance to the dentate line are independent factors for defecation dysfunction.