Distinct roles of excitatory and inhibitory neurons in the medial prefrontal cortex in the expression and reconsolidation of methamphetamine-associated memory in male mice.

Methamphetamine, a commonly abused drug, is known for its high relapse rate. The persistence of addictive memories associated with methamphetamine poses a significant challenge in preventing relapse. Memory retrieval and subsequent reconsolidation provide an opportunity to disrupt addictive memories. However, the key node in the brain network involved in methamphetamine-associated memory retrieval has not been clearly defined. In this study, using the conditioned place preference in male mice, whole brain c-FOS mapping and functional connectivity analysis, together with chemogenetic manipulations of neural circuits, we identified the medial prefrontal cortex (mPFC) as a critical hub that integrates inputs from the retrosplenial cortex and the ventral tegmental area to support both the expression and reconsolidation of methamphetamine-associated memory during its retrieval. Surprisingly, with further cell-type specific analysis and manipulation, we also observed that methamphetamine-associated memory retrieval activated inhibitory neurons in the mPFC to facilitate memory reconsolidation, while suppressing excitatory neurons to aid memory expression. These findings provide novel insights into the neural circuits and cellular mechanisms involved in the retrieval process of addictive memories. They suggest that targeting the balance between excitation and inhibition in the mPFC during memory retrieval could be a promising treatment strategy to prevent relapse in methamphetamine addiction.

MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway as a critical route affecting the toxic effects of lead on HK-2 cells.

Lead is a widespread environmental pollutant with serious adverse effects on human health, but the mechanism underlying its toxicity remains elusive. This study aimed to investigate the role of miR-584-5p / Ykt6 axis in the toxic effect of lead on HK-2 cells and the related mechanism. Our data suggested that lead exposure caused significant cytotoxicity, DNA and chromosome damage to HK-2 cells. Mechanistically, lead exposure down-regulated miR-584-5p and up-regulated Ykt6 expression, consequently, autophagosomal number and autophagic flux increased, lysosomal number and activity decreased, exosomal secretion increased. Interestingly, when miR-584-5p level was enhanced with mimic, autophagosomal number and autophagic flux decreased, lysosomal number and activity increased, ultimately, exosomal secretion was down-regulated, which resulted in significant aggravated toxic effects of lead. Further, directly blocking exosomal secretion with inhibitor GW4869 also resulted in exacerbated toxic effects of lead. Herein, we conclude that miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway may be a critical route affecting the toxic effects of lead on HK-2 cells. We provide a novel insight into the mechanism underlying the toxicity of lead on human cells.

Molecular epidemiology and virulence of goose astroviruses genotype-2 with different internal gene sequences.

Goose astrovirus (GAstV) is a small, non-enveloped, single-stranded, positive-sense RNA virus. GAstV has rapidly spread across various regions in China since 2016. In Sichuan, out of 113 samples were collected from goose diseases between 2019 and 2022, 97 were positive for GAstV through PCR testing. Remarkably, over the past three years, GAstV outbreak in Sichuan has accounted for an astonishing 85.8% of all goose-origin viruses. Among these cases, 63.9% had single GAstV infections, 29.9% had dual infections, and 6.2% had quadruple infections. To comprehend the variations in virulence among distinct strains of GAstV. 12 representative strains of single GAstV infections were isolated. These strains exhibited distinct characteristics, such as prominent white urate depositions in organs and joints, as well as extensive tissues phagocytosis in major target organs' tissues. The conserved ORF1b genes and the variable ORF2 genes of these representative GAstV strains were sequenced, enabling the establishment of phylogenetic trees for GAstV. All GAstV strains were identified as belonging to genotype-2 with varying internal gene sequences. Experiments were conducted on GAstV genotype-2, both in vivo and in vitro, revealed significant variations in pathogenicity and virulence across susceptible cells, embryos, and goslings. This comprehensive study enhances researchers' understanding of the transmission characteristics and virulence of GAstV genotype-2, aiding in a better comprehension of their molecular epidemiology and pathogenic mechanism.

mGluR5 in amygdala modulates fear memory generalization.

Introduction: Fear memory generalization is regarded as the core characteristic of posttraumatic stress disorder (PTSD) development. However, the mechanism that contributes to the generalization of conditioned fear memory is still unclear. The generalization is generally considered to be a mismatch that occurs during memory consolidation. Methods: Foot shocks and tones were given as unconditioned stress and conditioned stress, respectively for fear conditioning training. Immunofluorescence staining, western blotting and qPCR were performed to determine the expression of different genes in amygdala of mice after fear conditioning training. Cycloheximide was used as a protein synthesis inhibitor and 2-methyl-6-phenylethynyl-pyridine was injected for mGluR5 inhibition. Results: Fear conditioning using caused incremental generalization, which was clearly observed during training. The density of c-Fos+ cells or the synaptic p-NMDAR expression did not differ with stress intensities. Strong-shock fear conditioning could induce significant mGluR5 de novo synthesis in the amygdala, which was not observed in the weak-shock group. Inhibition of mGluR5 impaired fear memory generalization induced by strong-shock fear conditioning, but the generalization level induced by weak-shock training was enhanced. Discussion: These results indicated that mGluR5 in the amygdala is critical to the function of inappropriate fear memory generalization and suggested that this may be a potential target for the treatment of PTSD.

Genes and pathways associated with fear discrimination identified by genome-wide DNA methylation and RNA-seq analyses in nucleus accumbens in mice.

Fear memory is critical for individual survival. However, the maladaptive fear response is one of the hallmarks of fear-related disorders, which is characterized by the failure to discriminate threatening signals from neutral or safe cues. The biological mechanisms of fear discrimination remain to be clarified. In this study, we found that the nucleus accumbens (NAc) was indispensable for the formation of cued fear memory in mice, during which the expression of DNA methyltransferase 3a gene (DNMT3a) increased. Injection of Zebularine, a nonspecific DNMT inhibitor, into NAc immediately after conditioning induced a maladaptive fear response to neutral cue (CS-). Using whole-genome bisulfite sequencing (WGBS), differentially methylated sites and methylated regions (DMRs) were investigated. 16,226 DMRs in the genenome were identified, in which, 214 genes with significant differences in their methylation levels and mRNA expression profiles were identified through correlation analysis. Notably, 15 genes were synaptic function-related and 8 genes were enriched in the cGMP-PKG signaling pathway. Moreover, inhibition of PKG impaired fear discrimination. Together, our results revealed the profile and role of genome-wide DNA methylation in NAc in the regulation of fear discrimination.

Ocular phenotype related SNP analysis in Southern Han Chinese population from Guangdong province.

Ocular phenotype is recognizable among Asians, including eyelid fold, fissure inclination, and canthal index. Here we screened 27 facial phenotype-associated SNPs and reported a preliminary study in 246 Chinese individuals of Han origin in Guangdong province. Results showed that rs17760296 could explain 6.2% of the eyelid fold variation and double eyelids were more likely to appear when one's genotype was TT. With respect to the canthal index, rs4791774 and rs642961 were significantly associated with it. However, no individual SNP was associated with fissure inclination. We further constructed two models to predict eyelid fold and canthal index and evaluated them with receiver operating characteristic (ROC) curves and support vector machine (SVM) regression, respectively. The models showed a moderate-to-high predictive capacity (AUC = 0.75, sensitivity = 76%, and specificity = 72%) for the eyelid fold while a mild performance (R2 = 0.1074, MSE = 0.0005, P-value = 0.024) for the canthal index. In conclusion, our study indicates that rs17760296 could be selected into the facial phenotype prediction system for the Southern Han Chinese population. More SNPs are encouraged to improve the prediction accuracy of the canthal index besides rs4791774 and rs642961.

Ecological and health risk assessment of heavy metals in soil and Chinese herbal medicines.

As medicinal plants can accumulate harmful metals from the native soil, people's consumption of these materials may cause the human body to accumulate toxic metal elements. This has given rise to people's concerns about the quality and safety of Chinese medicinal materials. This research aims to determine the levels of Cr, Ni, Cu, Zn, As, Cd, Hg and Pb in four medicinal plant species (Aster tataricus L.f., Salvia miltiorrhiza Bge, Radix Aucklandiae, Scutellaria baicalensis Georgi) and their native soil. All samples were collected from Qian'an city, beside Yanshan Mountain Range in Tangshan city, east Hebei Province, north China. The contents of heavy metals we detected in the soil conformed to the current limits. However, the Cd and Hg in the soil had a very high potential ecological risk because of their contents higher than the base level of local soil. The contents of Cu, Cd, Hg and Pb in some medicinal herbs exceeded the standards. The content of Cu in Radix Aucklandiae exceeded the standard by 3 times, and others exceeded the standard by less than one time. The comprehensive health risk assessment of heavy metals with chronic non-carcinogenic effects for human body showed that none of the four medicinal herbs can create a health risk. Thus, there is no strong positive correlation between heavy metal pollution in medicinal herbs and that in the native soil. Further research should be investigated to the connection between the heavy metal levels in the soil and plants, and the comprehensive effects of soil, air and irrigation water on heavy metal pollution of Chinese herbal medicines. We also recommend that Chinese herbal medicines should be cultivated and gathered only from controlled or uncontaminated areas.

Clinicopathological characteristics and outcomes of 23 patients with secretory carcinoma of major salivary glands.

This retrospective study investigated the clinicopathological characteristics of secretory carcinoma of salivary glands (SCSG) in 23 patients with histopathologically confirmed SCSG between January 2010 and December 2020. In total, 13 males and 10 females (ratio, 1.3:1) aged 10 - 69 years (median, 45 years) were enrolled in this study; the average disease duration was 2.44 years (0.25-20 years). Twenty-one patients (91.3%) had SCSG in the parotid gland, and two (8.7%) in the submandibular gland. All patients had single nodules of diameters 0.8-4.8 cm (average 2.6 cm); five with lymph node metastases, and two with distant metastases. Immunohistochemically, tumors stained positive for S-100, mammaglobin, CK7, GATA3 and pan-Trk, and negative for DOG1, P63, and calponin, with Ki-67 positivity from 1 to 50%. ETV6 gene rearrangement was confirmed in 15 patients. All patients underwent oncological resection, four had radioactive particles implanted postoperatively, one received chemotherapy, and seven underwent chemoradiotherapy. Six patients had regional recurrences, two distant metastases, and one died before the last follow-up. SCSGs are typically indolent, with a low locoregional recurrence rate and excellent survival. Prognosis is correlated to clinical stage, pathological grade, and surgical procedures.

Carbazole/Benzimidazole-Based Bipolar Molecules as the Hosts for Phosphorescent and Thermally Activated Delayed Fluorescence Emitters for Efficient OLEDs.

A series of carbazole/benzimidazole-based molecules, namely, o-CbzBiz, m-CbzBiz, and p-CbzBiz, were readily synthesized in three steps by integrating carbazole with benzimidazole via the ortho-, meta-, and para-positions of phenyl linked to N-phenyl carbazole. These bipolar molecules exhibited a maximum UV absorption band ranging from 310 to 327 nm and a maximum emission band ranging from 380 to 400 nm. Density functional theory calculations showed that the twist angles between the donor and acceptor moieties of these molecules were from 54.9 to 67.1°. Such a twisted structure hampered the π-electron conjugation within the molecule and resulted in high-lying LUMO levels and triplet energies, which make them suitable to be applied as host materials in OLED devices. Our results showed that a maximum external quantum efficiency (EQE) of OLED reached 21.8% when p-CbzBiz was applied as the host of a green phosphorescent emitter, i.e., Ir(ppy)2(acac). In addition, a maximum EQE of OLED reached 16.7% when o-CbzBiz with the host of a green TADF emitter, i.e., 4CzIPN. Moreover, these devices exhibited lower efficiency roll-off than the CBP-hosted device using the same emitters, which demonstrated the bipolar charge carrier property of carbazole/benzimidazole-based molecules.

Metabolic reprogramming of normal oral fibroblasts correlated with increased glycolytic metabolism of oral squamous cell carcinoma and precedes their activation into carcinoma associated fibroblasts.

Cancers show a metabolic shift towards aerobic glycolysis. By "corrupting" their microenvironment, carcinoma cells are able to obtain energy substrates to "fuel" their mitochondrial metabolism and cell growth in an autophagy-associated, paracrine manner. However, the metabolic changes and role of normal fibroblasts in this process remain unclear. We devised a novel, indirect co-culture system to elucidate the mechanisms of metabolic coupling between stromal cells and oral squamous cell carcinoma (OSCC) cells. Here, we showed that normal oral fibroblasts (NOFs) and OSCC become metabolically coupled through several processes before acquiring an activated phenotype and without inducing senescence. We observed, for the first time, that NOFs export mitochondria towards OSCCs through both direct contact and via indirect mechanisms. NOFs are activated and are able to acquire a cancer-associated fibroblasts metabolic phenotype when co-cultivation with OSSC cells, by undergoing aerobic glycolysis, secreting more reactive oxygen species (ROS), high L-lactate and overexpressing lactate exporter MCT-4, leading to mitochondrial permeability transition pore (mPTP) opening, hypoxia, and mitophagy. On the other hand, Cav-1-low NOFs generate L-lactate to "fuel" mitochondrial metabolism and anabolic growth of OSCC. Most interestingly, the decrease in AMPK activity and PGC-1α expression might involve in regulation of ROS that functions to maintain final energy and metabolic homeostasis. This indicated, for the first time, the existence of ATP and ROS homeostasis during carcinogenesis. Our study suggests that an efficient therapeutical approach has to target the multiple mechanisms used by them to corrupt the normal surrounding stroma and metabolic homeostasis.

Fibroblasts rescue oral squamous cancer cell from metformin-induced apoptosis via alleviating metabolic disbalance and inhibiting AMPK pathway.

Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Growing evidence suggests that it may exert antitumor effects in vivo and in vitro. However, even with the promising potency on defeating cancer cells, the pre-clinical and epidemiological studies of metformin on various kinds of cancers are not satisfactory, and the reasons and underlying mechanisms remain unknown. Since cancer is a complex system, dependent on a promoting microenvironment, we hypothesize that the interactions between cancer cells and their neighborhood fibroblasts are essential for metformin resistance. To test this, we used a cell co-culture model closely mimicking the in vivo interactions and metabolic exchanges between normal stromal cells (NOFs) and oral squamous cancer cells (OSCC). Here we show that while metformin can significantly inhibit cell growth and induce apoptosis of OSCC cultured alone in a dose-dependent manner through activating p-AMPKT172 and modulating Bcl-2, Bax, and cleaved PARP. However, when OSCC are co-cultured with NOFs the metformin effects on OSCC cells are annihilated. NOFs are rescuing OSCC from metformin - induced apoptosis, at least partially, through inhibiting the activity of AMPK and PARP, maintaining mitochondrial membrane potential and increasing the oxidative stress. Our results indicate that metformin effects on oral cancer cells are modulated by the microenvironment and that this has to be taken into consideration in the context of developing a new combination of drugs for oral cancer treatment.

Factors to Consider When Deciding on the Type of Free-Flap Reconstruction of Head and Neck Soft Tissue Defects.

OBJECTIVES: The aim of the study was to evaluate the functional and aesthetic outcomes on donor and recipient sites and to determine the effects of technical factors including flap thickness and vessel diameters measured by ultrasonography as well as the size of the defect and postoperative volume reduction of the flaps measured by magnetic resonance imaging (MRI). METHODS: In patients who had undergone soft tissue reconstructive surgery between March 2013 and March 2016 using 55 anterolateral thigh flaps (ALTFs), 30 radial forearm flaps (RFFs), and 18 latissimus dorsi flaps (LDFs), color Doppler ultrasonography was performed to measure the thickness of the flap at the site of the perforator. Preoperative color Doppler ultrasound examinations of the blood vessel diameters of donor and recipient sites were carried out. RESULTS: 97.1% of flaps showed complete survival and 2.9% complete failure (2 ALTFs and 1 LDF). The difference in flap volume of ALTFs, RFFs, and LDFs between MRI 1 (3-6 weeks) and MRI 2 (6-18 months) was 27.6, 17.9, and 36.1%, respectively. CONCLUSION: Proper selection of the flap is important for the optimization of the aesthetic and functional outcomes. Ultrasound, the surgeon's experience and the extension and nature of the defect play a key role in the selection of the flap.

Myoepithelial Carcinoma of the Salivary Glands: A Clinicopathologic Study of 29 Patients.

PURPOSE: Myoepithelial carcinomas (MECs) of the salivary glands are relatively rare. The clinicopathologic features, immunohistochemical profile, and biologic behavior have not been well-defined. MATERIALS AND METHODS: A total of 29 patients with MEC diagnosed during a 10-year period were included in the present study focusing on the biologic behavior, and the pathologic samples of 28 patients were collected for additional investigation of the histologic characteristics. Thirteen samples with detailed immunohistochemical results were included for illustrating immunohistochemical profiles. RESULTS: The parotid gland (n = 7) was the most common site involved, followed by the palate (n = 6) and the submandibular gland (n = 6). A multinodular growth pattern (n = 14) and sheet-like arrangement of tumor cells (n = 14) were observed. Of the 28 MEC samples, 14 (50%) were epithelioid, 5 (18%) were clear cell, 5 (18%) were plasmacytoid, 3 (11%) were mixed cell type, and 1 (3%) was spindle. The tumor-associated matrix was more prevalently hyalinized than myxoid. Of the 28 cases, 12 (43%) were classified as high grade and 16 (57%) as low grade. Immunohistochemical analysis revealed pan-cytokeratin (92.3%), smooth muscle actin (36%), S-100 protein (54.5%), and p63 (91.7%) positivity and carcinoembryonic antigen (100%) negativity. Ki-67 was immunoreactive in 62% of the MECs, with the Ki-67 labeling index ranging from less than 5 to 20%. Eleven patients developed recurrence (median disease-free survival 43 months) and 11 (44%) developed metastases. Two patients (8%) died of disease after a mean period of 18 months. Fourteen patients (61%) were without any evidence of disease after a mean of 32.5 months (range 3 to 86). The mitotic rate correlated weakly (P = .042) with a poor outcome, but none of the other factors showed a significant correlation with the prognosis. CONCLUSION: MECs of the salivary glands have a relatively high recurrence rate and metastasis rate and a long period of survival with tumor. A combination of pathologic features and various immunohistochemical indexes are crucial for the accurate diagnosis of MECs. Extensive excision is the favorable choice for treating MECs, and suprahyoid lymph node dissection is recommended when the submandibular gland is involved.

Hepatotoxicity and nephrotoxicity of organic contaminants in wastewater-irrigated soil.

The objective of this study is to investigate the hepatotoxicity and nephrotoxicity of organic contaminants in wastewater-irrigated soil using in vivo and in vitro experiments on mice and rat. Soil samples were collected from a wastewater-irrigated area and groundwater-irrigated area, i.e. clean water-irrigated area as control group. The organic contaminants were extracted using an ultrasonic oscillator. In vivo experiment was performed by contamination of hepatocytes of rat using the organic extract, and comet assay was used to analyse the DNA damage of hepatocytes. For in vitro experiment, mice were first gavaged with extracts, and then the indicators for kidney functions, liver functions and oxidative damage of tissues were investigated. The result shows, for in vitro experiments, compared with clean water-irrigated area groups, the average DNA tailing length for the wastewater-irrigated area group is larger, and for the wastewater-irrigated area groups with extract concentration 0.6 g/ml and 0.9 g/ml, the tailing rate increases significantly (P < 0.05). For in vivo experiments, the change of weight across each group shows no significant difference (P < 0.05). Compared with clean water-irrigated groups, the liver indices have decreased for all groups of the wastewater-irrigated area, while both kidney and liver indices decreased for wastewater-irrigated area high-dose group (P < 0.05 or P < 0.01). The total proteins for wastewater-irrigated low-dose group and Gamma-glutamyl transpeptidase, creatinine for high-dose group all increased (P < 0.01). Compared with the reagent control group, total superoxide dismutase activity of liver for wastewater-irrigated groups and glutathione peroxidase activity for high-dose group, malondialdehyde content all decreased (P < 0.05 or P < 0.01); glutathione peroxidase activity of kidney tissue for wastewater-irrigated high-dose group decreased (P < 0.01). The result shows that the joint toxicity in extracts of wastewater-irrigated soil is able to cause DNA damage of hepatocytes in rats, changes of liver functions in mice and lead to oxidative damage of liver and kidney.