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Phthalate esters (PAEs) are an emerging pollutant due to widespread distribution in environmental mediums that have attracted widespread attention over recent years. However, there is little information about tea plantation soil PAEs. A total of 270 soil samples collected from 45 tea plantations in the major high-quality tea-producing regions of Jiangsu, Zhejiang, and Anhui provinces in China were analyzed for seven PAEs. The detection frequency of PAEs in tea plantation soil was 100%. DBP, DEHP, and DiBP were the main congeners in tea plantation soil. The PAEs concentrations in the upper soil were significantly higher than those in the lower soil. The concentration of tea plantation soil PAEs in Jiangsu Province was significantly lower than those in Zhejiang and Anhui provinces. Intercropping with chestnuts can effectively reduce the contamination level of PAEs in tea plantation soil. Correlation analysis, redundancy analysis, partial correlation analysis, and structural equation modeling methods further confirmed the strong direct influence of factors such as chestnut–tea intercropping, temperature, and agricultural chemicals on the variation of PAEs in tea plantation soil. The health and ecological risk assessments indicated that non-carcinogenic risk was within a safe range and that there was a high carcinogenic risk via the dietary pathway, with DBP posing the highest ecological risk.

Abstract Background Whether programmed cell death-1/ligand-1 (PD-1/PD-L1) blockade-based neoadjuvant treatment may benefit locally advanced oncogene-mutant non–small cell lung cancer (NSCLC) patients remains controversial. This retrospective study was designed to observe the efficacy and safety of neoadjuvant PD-1/PD-L1 blockade plus chemotherapy versus chemotherapy and corresponding tyrosine kinase inhibitors (TKIs) in patients with resectable oncogene-positive NSCLC. Methods Patients with potential resectable NSCLC harbouring oncogene alterations who had received neoadjuvant treatment were retrospectively recruited, and an oncogene-negative cohort of patients who received neoadjuvant PD-(L)1 blockade-based neoadjuvant treatment was reviewed for comparison during the same period. The primary aim was to observe the treatment efficacy and event-free survival (EFS) of these agents. Safety profile, molecular target, and immunologic factor data, including PD-L1 expression and tumour mutational burden (TMB), were also obtained. Results A total of 46 patients were recruited. Thirty-one of them harboured oncogene alterations, including EGFR, KRAS, ERBB2, ROS1, MET, RET, ALK, and FGFR3 alterations. Among the oncogene-positive patients, 18 patients received neoadjuvant PD-(L)1 blockade immunotherapy plus chemotherapy (oncogene-positive IO group), 13 patients were treated with neoadjuvant chemotherapy and/or corresponding TKIs or TKIs alone (oncogene-positive chemo/TKIs group), and the other 15 patients were oncogene negative and received neoadjuvant PD-(L)1 blockade plus chemotherapy (oncogene-negative IO group). The pathological complete response (pCR) and major pathological response (MPR) rates were 22.2% (4 of 18) and 44.4% (8 of 18) in the oncogene-positive IO group, 0% (P = 0.120) and 23.1% (3 of 13) (P = 0.276) in the oncogene-positive chemo/TKIs group, and 46.7% (7 of 15) (P = 0.163) and 80.0% (12 of 15) (P = 0.072) in the oncogene-negative IO group, respectively. By the last follow-up, the median EFS time had not reached in the oncogene-positive IO group, and was 29.5 months in the oncogene-positive chemo/TKIs group and 38.4 months in the oncogene-negative IO group. Conclusion Compared with chemotherapy/TKIs treatment, neoadjuvant treatment with PD-(L)1 blockade plus platinum-based chemotherapy was associated with higher pCR/MPR rates in patients with partially resectable oncogene-mutant NSCLC, while the pCR/MPR rates were lower than their oncogene-negative counterparts treated with PD-(L)1 blockade-based treatment. Specifically, oncogene alteration types and other predictors of response to immunotherapy should be taken into account in clinical practice.

Abstract The gold standard for enrollment and endpoint assessment in metabolic dysfunction‐associated steatosis clinical trials is histologic assessment of a liver biopsy performed on glass slides. However, obtaining the evaluations from several expert pathologists on glass is challenging, as shipping the slides around the country or around the world is time‐consuming and comes with the hazards of slide breakage. This study demonstrated that pathologic assessment of disease activity in steatohepatitis, performed using digital images on the AISight whole slide image management system, yields results that are comparable to those obtained using glass slides. The accuracy of scoring for steatohepatitis (nonalcoholic fatty liver disease activity score ≥4 with ≥1 for each feature and absence of atypical features suggestive of other liver disease) performed on the system was evaluated against scoring conducted on glass slides. Both methods were assessed for overall percent agreement with a consensus “ground truth” score (defined as the median score of a panel of three pathologists’ glass slides). Each case was also read by three different pathologists, once on glass and once digitally with a minimum 2‐week washout period between the modalities. It was demonstrated that the average agreement across three pathologists of digital scoring with ground truth was noninferior to the average agreement of glass scoring with ground truth [noninferiority margin: −0.05; difference: −0.001; 95% CI: (−0.027, 0.026); and p

Abstract Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.

Objective: FAS-mediated apoptosis of hepatocytes and aberrant TGF-β signaling are major drivers of liver fibrosis. Decreased miRNA let-7 expression in the livers of patients and animals with fibrosis suggests a mechanistic link of let-7 to hepatic fibrogenesis. Methods: Using transient transfection we tested the effects of let-7 overexpression and TET3 siRNA knockdown on FAS and TGF-β1 expression and FAS-mediated apoptosis in human and mouse primary hepatocytes. We assessed the therapeutic activity of let-7 miRNA delivered via adeno-associated viral vectors in mouse models of carbon tetrachloride (CCl4)-induced and bile duct ligation (BDL)-induced liver fibrosis. Results: Let-7 decreased TGF-β1 production from hepatocytes through a negative feedback loop involving TET3. On the other hand, let-7 post-transcriptionally inhibits FAS expression, thereby suppressing hepatocyte apoptosis. Hepatic-specific delivery of let-7 miRNA mitigated liver fibrosis in both CCl4 and BDL mouse models. Conclusions: Let-7 is a crucial node in the signaling networks that govern liver fibrosis progression. Let-7 and/or its derivatives may be used as therapeutic agents for liver fibrosis.

Abstract Background Circumferential resection margin (CRM) is very important in esophageal cancer, but its diagnostic criteria has not been unified. The College of American Pathologists (CAP) and the Royal College of Pathologists (RCP) provide two different criteria. The aim of this study is to evaluate the long-term prognostic significance of CRM status with different CRM criteria in esophageal squamous cell carcinoma (ESCC). Methods Influence of CRM status according to the CAP and RCP criteria on long-term survival of 838 patients with resected pT3 tumors and without neoadjuvant therapy was analyzed. Patients stratified into three groups on the basis of tumor distance from the CRM (CRM > 1 mm, 0-1 mm, and 0 mm) were also analysed. Results Positive CRM was found in 59 (7%) patients according to the CAP criteria and 317 (37.8%) patients according to the RCP criteria. Univariate and multivariate survival analysis showed that CRM status, according to three different criteria, was independent prognostic factor. However, subgroup analysis showed that the prognostic value of CRM status was limited to certain metastatic lymph node load. In pN0 subgroup, patients with CRM > 1 mm had better prognosis than patients with CRM 0-1 mm. Patients with CRM 0 mm had worse outcome than patients with CRM > 0 mm in pN1-2 subgroup. But CRM status had no prognosis value in pN3 subgroup. Conclusions The CRM status is an important prognostic factor in ESCC patients, but this effect was limited to patients without or with less lymph node metastasis (pN0-2). In clinical practice, we recommend the 1 mm-three-tier criteria as it provides more prognostic value than the traditional two-tier criteria.

Background: PD-1 blockade has been shown to have promising efficacy and acceptable safety profiles in advanced and metastatic gastric cancer; however, the efficacy and safety of neoadjuvant PD-1 blockade-based immunotherapy plus chemotherapy in locally advanced gastric cancer (LAGC) remain uncertain. Methods: We performed a retrospective review of patients with LAGC who received neoadjuvant treatment followed by D2 radical resection at the Affiliated Hospital of Qingdao University from 2019 to 2021. The primary aim was to investigate the difference in pathological response rates between neoadjuvant PD-1 immunotherapy plus chemotherapy and neoadjuvant chemotherapy alone. Multivariable models for pathological complete response (pCR) were constructed to investigate the factors that facilitate pCR. Trial registration: QYFYWZLL27406. Results: A total of 77 patients were included in the analysis, among whom 34 (44.2%) received neoadjuvant PD-1 blockade immunotherapy plus chemotherapy. A higher pCR rate was observed in the neoadjuvant PD-1 blockade immunotherapy plus chemotherapy group (8 of 34, 23.5% vs. 2 of 43, 4.7%, P=0.019). Multivariate logistic regression analysis of pCR revealed neoadjuvant PD-1 blockade plus chemotherapy regimen promoted pCR (OR 12.95, P=0.016). Regarding safety, 76.5% (26 of 34) of patients in the PD-1 blockade plus chemotherapy group and 76.7% (33 of 43) of patients in the chemotherapy group experienced treatment-related adverse events (TRAEs), and grade 3 or worse adverse events were 29.4% (10 of 34) and 34.9% (15 of 43), respectively. Conclusion: Neoadjuvant PD-1 blockade plus chemotherapy induced a higher pCR rate than neoadjuvant chemotherapy, and the combined therapy was tolerable in LAGC patients.

Animals can continuously learn different tasks to adapt to changing environments and, therefore, have strategies to effectively cope with inter-task interference, including both proactive interference (Pro-I) and retroactive interference (Retro-I). Many biological mechanisms are known to contribute to learning, memory, and forgetting for a single task, however, mechanisms involved only when learning sequential different tasks are relatively poorly understood. Here, we dissect the respective molecular mechanisms of Pro-I and Retro-I between two consecutive associative learning tasks in Drosophila. Pro-I is more sensitive to an inter-task interval (ITI) than Retro-I. They occur together at short ITI (

Abstract Extensive studies concluded that homophilic interactions between pre- and postsynaptic teneurins, evolutionarily conserved cell-adhesion molecules, encode the specificity of synaptic connections. However, no direct evidence is available to demonstrate that teneurins are actually required on both pre- and postsynaptic neurons for establishing synaptic connections, nor is it known whether teneurins are localized to synapses. Using super-resolution microscopy, we demonstrate that Teneurin-3 assembles into presynaptic nanoclusters of approximately 80 nm in most excitatory synapses of the hippocampus. Presynaptic deletions of Teneurin-3 and Teneurin-4 in the medial entorhinal cortex revealed that they are required for assembly of entorhinal cortex-CA1, entorhinal cortex-subiculum, and entorhinal cortex-dentate gyrus synapses. Postsynaptic deletions of teneurins in the CA1 region, however, had no effect on synaptic connections from any presynaptic input. Our data suggest that different from the current prevailing view, teneurins promote the establishment of synaptic connections exclusively as presynaptic cell-adhesion molecules, most likely via their nanomolar-affinity binding to postsynaptic latrophilins.

Treatment for liver cancer is complicated by its various subtypes, which show different responses to anticancer drugs. This study demonstrates the effectiveness of targeting biophysical phenotypes related to cytoskeleton properties that are usually masked in traditional drug screens.

Summary: Oncogenic mutations in the EGFR gene account for 15–20% of lung adenocarcinoma (LUAD) cases. However, the mechanism for EGFR driven tumor development and growth is not fully understood. Here, using an mRNA expression profiling-based approach we identified betacellulin (BTC) as one the gene upregulated by oncogenic EGFR in an MAP kinase-dependent manner. BTC protein expression was markedly increased in LUAD patient samples compared to normal lung tissue, with higher expression in EGFR-mutant LUAD. BTC was sufficient to transform immortalized mouse cells, initiate tumor development in mice, and promote the survival of immortalized human lung epithelial cells. Conversely, knockdown of BTC inhibited the growth of EGFR-mutant human LUAD cells in culture and their tumor-forming ability in mice. Mechanistically, BTC knockdown resulted in attenuated EGFR signaling and apoptosis induction. Collectively, these results demonstrate a key role of BTC in EGFR-mutant LUAD, with potential therapeutic implications in LUAD and other EGFR-mutant cancers.

Abstract In preclinical research, histological analysis of tissue samples is often limited to qualitative or semiquantitative scoring assessments. The reliability of this analysis can be impaired by the subjectivity of these approaches, even when read by experienced pathologists. Furthermore, the laborious nature of manual image assessments often leads to the analysis being restricted to a relatively small number of images that may not accurately represent the whole sample. Thus, there is a clear need for automated image analysis tools that can provide robust and rapid quantification of histologic samples from paraffin‐embedded or cryopreserved tissues. To address this need, we have developed a color image analysis algorithm (DigiPath) to quantify distinct color features in histologic sections. We demonstrate the utility of this tool across multiple types of tissue samples and pathologic features, and compare results from our program to other quantitative approaches such as color thresholding and hand tracing. We believe this tool will enable more thorough and reliable characterization of histological samples to facilitate better rigor and reproducibility in tissue‐based analyses.

Objective: Metabolic deregulation is a key hallmark of cancer cells and has been shown to drive cancer growth and metastasis. However, not all metabolic drivers of melanoma are known. Based on our finding that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in melanoma, the objective of these studies was to establish its role in melanoma tumor growth and metastasis, understand its mechanism of action, and evaluate ASAH1 targeting for melanoma therapy. Methods: We used publicly available melanoma datasets and patient-derived samples of melanoma and normal skin tissue and analyzed them for ASAH1 mRNA expression and ASAH1 protein expression using immunohistochemistry. ASAH1 was knocked down using short-hairpin RNAs in multiple melanoma cell lines that were tested in a series of cell culture-based assays and mouse-based melanoma xenograft assays to monitor the effect of ASAH1 knockdown on melanoma tumor growth and metastasis. An unbiased metabolomics analysis was performed to identify the mechanism of ASAH1 action. Based on the metabolomics findings, the role of peroxisome-mediated reactive oxygen species (ROS) production was explored in regard to mediating the effect of ASAH1. The ASAH1 inhibitor was used alone or in combination with a BRAFV600E inhibitor to evaluate the therapeutic value of ASAH1 targeting for melanoma therapy. Results: We determined that ASAH1 was overexpressed in a large percentage of melanoma cells and regulated by transcription factor E2F1 in a mitogen-activated protein (MAP) kinase pathway-dependent manner. ASAH1 expression was necessary to maintain melanoma tumor growth and metastatic attributes in cell cultures and mouse models of melanoma tumor growth and metastasis. To identify the mechanism by which ASAH1 facilitates melanoma tumor growth and metastasis, we performed a large-scale and unbiased metabolomics analysis of melanoma cells expressing ASAH1 short-hairpin RNAs (shRNAs). We found that ASAH1 inhibition increased peroxisome biogenesis through ceramide-mediated PPARγ activation. ASAH1 loss increased ceramide and peroxisome-derived ROS, which in turn inhibited melanoma growth. Pharmacological inhibition of ASAH1 also attenuated melanoma growth and enhanced the effectiveness of BRAF kinase inhibitor in the cell cultures and mice. Conclusions: Collectively, these results demonstrate that ASAH1 is a druggable driver of melanoma tumor growth and metastasis that functions by suppressing peroxisome biogenesis, thereby inhibiting peroxisome-derived ROS production. These studies also highlight the therapeutic utility of ASAH1 inhibitors for melanoma therapy.

New protein synthesis is known to be indispensable for the consolidation of long-term memory. Here, the authors report that an olfactory memory can be successfully recalled after 14 days without protein synthesis when the training context is also provided in addition to the conditioned odor.

Summary: Pathological activation of TGF-β signaling is universal in fibrosis. Aberrant TGF-β signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-β pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-β1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-β1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases. : Xu et al. unmask a positive feedback loop between chromatin demethylase TET3 and TGF-β1 in stressed hepatocytes and stellate cells in humans and mice. Activation of this loop stimulates expression of fibrotic genes, whereas knockdown of TET3 reduces liver fibrosis in mice, suggesting a strategy for treating fibrosis.

Summary: Multiple brain regions respond to harmful nociceptive stimuli. However, it remains unclear as to whether behavioral avoidance of such stimuli can be modulated within the same or distinct brain networks. Here, we found subgroups of neurons localized within a well-defined brain region capable of mediating both innate and conditioned nociceptive avoidance in Drosophila. Neurons in the ventral, but not the dorsal, of the multiple-layer organized fan-shaped body (FB) are responsive to electric shock (ES). Silencing ES-responsive neurons, but not non-responsive neurons, leads to reduced avoidance of harmful stimuli, including ES and heat shock. Activating these neurons consistently triggers avoidance and can serve as an unconditional stimulus in an aversive classical conditioning task. Among the three groups of responsive neurons identified, two also have reduced activity in ES-conditioned odor avoidance. These results demonstrate that both innate and conditioned nociceptive avoidance might be represented within neurons confined to a single brain region. : Regulation of innate and conditioned nociceptive avoidance is critical for animal survival. Hu et al. find that neurons in the fan-shaped body, a multiple-layer organized brain region, are highly responsive to harmful stimuli, including electric shock, and can regulate both innate and conditioned avoidance of such stimuli in Drosophila. Keywords: Drosophila, fan-shaped body, nociception, innate avoidance, conditioned avoidance

Repeated learning is used daily and is a powerful way to improve memory. A fundamental question is how multiple learning trials add up to improve memory. While the major studies so far of such a repetition effect have emphasized the strengthening of memory formation, the current study reveals a molecular mechanism through suppression of forgetting. We find that single-session training leads to formation of anesthesia-resistant memory (ARM) and then activation of the small G protein Cdc42 to cause decay or forgetting of ARM within 24 hr. Repetition suppresses the activation of Cdc42-dependent forgetting, instead of enhancing ARM formation, leading to prolonged ARM. Consistently, inhibition of Cdc42 activity through genetic manipulation mimicked the repetition effect, while repetition-induced ARM improvement was abolished by elevated Cdc42 activity. Thus, only the first session in repetitive training contributes to ARM formation, while the subsequent sessions are devoted not to acquiring information but to inhibiting forgetting.

Inhibition of immune checkpoint T cell regulatory molecules by using programmed cell death protein 1 (PD-1), or its ligand (PDL-1), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) has been increasingly used to treat advanced malignancies. The immune-related adverse effects associated with these treatments such as diarrhea, colitis, and CTLA-4 treatment-associated perforating colitis have been reported. However, anti-PD-1/PD-L1-associated perforating colitis has rarely been reported. We report a case of colonic perforation in a patient recently treated with pembrolizumab, a PD-1 inhibitor for metastatic melanoma. Awareness of anti-PD-1/PD-L1-associated colitis and perforation will facilitate a timely diagnosis and management as they are increasingly used in oncology.

Background & Aims: Pigment epithelium-derived factor (PEDF) is a secretory protein that inhibits multiple tumor types. PEDF inhibits the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (LRP6), in the eye, but whether the tumor-suppressive properties of PEDF occur in organs such as the liver is unknown. Methods: Wnt-dependent regulation of PEDF was assessed in the absence and presence of the Wnt coreceptor LRP6. Whole genome expression analysis was performed on PEDF knockout (KO) and control livers (7 months). Interrogation of Wnt/β-catenin signaling was performed in whole livers and human hepatocellular carcinoma (HCC) cell lines after RNA interference of PEDF and restoration of a PEDF-derived peptide. Western diet feeding for 6 to 8 months was used to evaluate whether the absence of PEDF was permissive for HCC formation (n = 12/group). Results: PEDF levels increased in response to canonical Wnt3a in an LRP6-dependent manner but were suppressed by noncanonical Wnt5a protein in an LRP6-independent manner. Gene set enrichment analysis (GSEA) of PEDF KO livers revealed induction of pathways associated with experimental and human HCC and a transcriptional profile characterized by Wnt/β-catenin activation. Enhanced Wnt/β-catenin signaling occurred in KO livers, and PEDF delivery in vivo reduced LRP6 activation. In human HCC cells, RNA interference of PEDF led to increased levels of activated LRP6 and β-catenin, and a PEDF 34-mer peptide decreased LRP6 activation and β-catenin signaling, and reduced Wnt target genes. PEDF KO mice fed a Western diet developed sporadic well-differentiated HCC. Human HCC specimens demonstrated decreased PEDF staining compared with hepatocytes. Conclusions: PEDF is an endogenous inhibitor of Wnt/β-catenin signaling in the liver. Keywords: Extracellular Matrix, PEDF, Wnt/β-Catenin