Characterization of network hierarchy reflects cell state specificity in genome organization.

Dynamic chromatin structure acts as the regulator of transcription program in crucial processes including cancer and cell development, but a unified framework for characterizing chromatin structural evolution remains to be established. Here, we performed graph inferences on Hi-C data sets and derived the chromatin contact networks. We discovered significant decreases in information transmission efficiencies in chromatin of colorectal cancer (CRC) and T-cell acute lymphoblastic leukemia (T-ALL) compared to corresponding normal controls through graph statistics. Using network embedding in the Poincaré disk, the hierarchy depths of chromatin from CRC and T-ALL patients were found to be significantly shallower compared to their normal controls. A reverse trend of change in chromatin structure was observed during early embryo development. We found tissue-specific conservation of hierarchy order in chromatin contact networks. Our findings reveal the top-down hierarchy of chromatin organization, which is significantly attenuated in cancer.

Diffusion-enhanced characterization of 3D chromatin structure reveals its linkage to gene regulatory networks and the interactome.

The interactome networks at the DNA, RNA, and protein levels are crucial for cellular functions, and the diverse variations of these networks are heavily involved in the establishment of different cell states. We have developed a diffusion-based method, Hi-C to geometry (CTG), to obtain reliable geometric information on the chromatin from Hi-C data. CTG produces a consistent and reproducible framework for the 3D genomic structure and provides a reliable and quantitative understanding of the alterations of genomic structures under different cellular conditions. The genomic structure yielded by CTG serves as an architectural blueprint of the dynamic gene regulatory network, based on which cell-specific correspondence between gene-gene and corresponding protein-protein physical interactions, as well as transcription correlation, is revealed. We also find that gene fusion events are significantly enriched between genes of short CTG distances and are thus close in 3D space. These findings indicate that 3D chromatin structure is at least partially correlated with downstream processes such as transcription, gene regulation, and even regulatory networking through affecting protein-protein interactions.

Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Activation of nicotinic acetylcholine receptor α7 subunit limits Zika viral infection via promoting autophagy and ferroptosis.

Vagus nerve regulates viral infection and inflammation via the alpha 7 nicotinic acetylcholine receptor (α7 nAChR); however, the role of α7 nAChR in ZIKA virus (ZIKV) infection, which can cause severe neurological diseases such as microcephaly and Guillain-Barré syndrome, remains unknown. Here, we first examined the role of α7 nAChR in ZIKV infection in vitro. A broad effect of α7 nAChR activation was identified in limiting ZIKV infection in multiple cell lines. Combined with transcriptomics analysis, we further demonstrated that α7 nAChR activation promoted autophagy and ferroptosis pathways to limit cellular ZIKV viral loads. Additionally, activation of α7 nAChR prevented ZIKV-induced p62 nucleus accumulation, which mediated an enhanced autophagy pathway. By regulating proteasome complex and an E3 ligase NEDD4, activation of α7 nAChR resulted in increased amount of cellular p62, which further enhanced the ferroptosis pathway to reduce ZIKV infection. Moreover, utilizing in vivo neonatal mouse models, we showed that α7 nAChR is essential in controlling the disease severity of ZIKV infection. Taken together, our findings identify an α7 nAChR-mediated effect that critically contributes to limiting ZIKV infection, and α7 nAChR activation offers a novel strategy for combating ZIKV infection and its complications.

Clinicopathologic Features of Gastrointestinal Tract Langerhans Cell Histiocytosis.

Gastrointestinal (GI) tract involvement by Langerhans cell histiocytosis (LCH) is rare and its clinicopathologic characteristics have only been described in case reports and small series. We reviewed hematoxylin and eosin and CD1a, S100, and Langerin immunohistochemical-stained slides from 47 patients with well-documented demographic and clinical findings. Our cases included 8 children and 39 adults, with a mean follow-up of 63 months. All pediatric patients had concurrent multisystem LCH, presented with GI symptoms, and showed nonpolypoid lesions. Seven (88%) showed multifocal GI disease, including 5 with multiple GI organ involvement. All sampled lesions from children exhibited infiltrative growth. More than half had died of the disease or manifested persistent LCH at last follow-up. Twenty-five of 39 (64%) adults had LCH involving only the GI tract (single system), with the remaining 14 (36%) exhibiting multisystem disease. Adult single-system GI LCH was typically encountered incidentally on screening/surveillance endoscopy (72%). Most exhibited isolated colorectal involvement (88%) as a solitary polyp (92%), with a well-demarcated/noninfiltrative growth pattern (70%), and excellent prognosis (100%). In comparison, adult patients with multisystem LCH more frequently presented with GI symptoms (92%, P < .001), noncolorectal GI site involvement (50%, P = .02), multifocal GI lesions (43%, P = .005), nonpolypoid lesions (71%, P < .001), infiltrative histologic growth pattern (78%, P = .04), and persistent disease (57%, P < .001). Adult patients with multisystem LCH appear to exhibit similar clinicopathologic features to those of pediatric patients. These results demonstrated that adults with single-system LCH involving the GI tract have an excellent prognosis, whereas multisystem LCH occurring at any age carries an unfavorable prognosis. High-risk features of GI LCH include pediatric age, GI symptomatology, noncolorectal GI involvement, multifocal GI disease, nonpolypoid lesions, and infiltrative growth pattern.

Subgrading of G2 Pancreatic Neuroendocrine Tumors as 2A (Ki67 3% to < 10%) Versus 2B (10% to ≤ 20%) Identifies Behaviorally Distinct Subsets in Keeping with the Evolving Management Protocols.

BACKGROUND: Grade 1/2 PanNETs are mostly managed similarly, typically without any adjunct treatment with the belief that their overall metastasis rate is low. In oncology literature, Ki67-index of 10% is increasingly being used as the cutoff in stratifying patients to different protocols, although there are no systematic pathology-based studies supporting this approach. METHODS: Ki67-index was correlated with clinicopathologic parameters in 190 resected PanNETs. A validation cohort (n = 145) was separately analyzed. RESULTS: In initial cohort, maximally selected rank statistics method revealed 12% to be the discriminatory cutoff (close to 10% rule of thumb). G2b cases had liver/distant metastasis rate of almost threefold higher than that of G2a and showed significantly higher frequency of all histopathologic signs of aggressiveness (tumor size, perineural/vascular invasion, infiltrative growth pattern, lymph node metastasis). In validation cohort, these figures were as striking. When all cases were analyzed together, compared with G1, the G2b category had nine times higher liver/distant metastasis rate (6.1 vs. 58.5%; p < 0.001) and three times higher lymph node metastasis rate (20.5 vs. 65.1%; p < 0.001). CONCLUSIONS: G2b PanNETs act very similar to G3, supporting management protocols that regard them as potential therapy candidates. Concerning local management, metastatic behavior in G2b cases indicate they may not be as amenable for conservative approaches, such as watchful waiting or enucleation. This substaging should be considered into diagnostic guidelines, and clinical trials need to be devised to determine the more appropriate management protocols for G2b (10% to ≤ 20%) group, which shows liver/distant metastasis in more than half of the cases, which at minimum warrants closer follow-up.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Steric repulsion introduced by loop constraints modulates the microphase separation of chromatins.

Within the confines of a densely populated cell nucleus, chromatin undergoes intricate folding, forming loops, domains, and compartments under the governance of topological constraints and phase separation. This coordinated process inevitably introduces interference between different folding strategies. In this study, we model interphase chromatins as block copolymers with hetero-hierarchical loops within a confined system. Employing dissipative particle dynamics simulations and scaling analysis, we aim to explain how the structure and distribution of loop domains modulate the microphase separation of chromatins. Our results highlight the correlation between the microphase separation of the copolymer and the length, heterogeneity, and hierarchically nested levels of the loop domains. This correlation arises from steric repulsion intrinsic to loop domains. The steric repulsion induces variations in chain stiffness (including local orientation correlations and the persistence length), thereby influencing the degree of phase separation. Through simulations of block copolymers with distinct groups of hetero-hierarchical loop anchors, we successfully reproduce changes in phase separation across diverse cell lines, under fixed interaction parameters. These findings, in qualitative alignment with Hi-C data, suggest that the variations of loop constraints alone possess the capacity to regulate higher-order structures and the gene expressions of interphase chromatins.

Co-exposure to polystyrene nanoplastics and triclosan induces synergistic cytotoxicity in human KGN granulosa cells by promoting reactive oxygen species accumulation.

In recent years, nanoplastics (NPs) and triclosan (TCS, a pharmaceutical and personal care product) have emerged as environmental pollution issues, and their combined presence has raised widespread concern regarding potential risks to organisms. However, the combined toxicity and mechanisms of NPs and TCS remain unclear. In this study, we investigated the toxic effects of polystyrene NPs and TCS and their mechanisms on KGN cells, a human ovarian granulosa cell line. We exposed KGN cells to NPs (150 µg/mL) and TCS (15 µM) alone or together for 24 hours. Co-exposure significantly reduced cell viability. Compared with exposure to NPs or TCS alone, co-exposure increased reactive oxygen species (ROS) production. Interestingly, co-exposure to NPs and TCS produced synergistic effects. We examined the activity of superoxide dismutase (SOD) and catalase (CAT), two antioxidant enzymes; it was significantly decreased after co-exposure. We also noted an increase in the lipid oxidation product malondialdehyde (MDA) after co-exposure. Furthermore, co-exposure to NPs and TCS had a more detrimental effect on mitochondrial function than the individual treatments. Co-exposure activated the NRF2-KEAP1-HO-1 antioxidant stress pathway. Surprisingly, the expression of SESTRIN2, an antioxidant protein, was inhibited by co-exposure treatments. Co-exposure to NPs and TCS significantly increased the autophagy-related proteins LC3B-II and LC3B-Ⅰ and decreased P62. Moreover, co-exposure enhanced CASPASE-3 expression and inhibited the BCL-2/BAX ratio. In summary, our study revealed the synergistic toxic effects of NPs and TCS in vitro exposure. Our findings provide insight into the toxic mechanisms associated with co-exposure to NPs and TCS to KGN cells by inducing oxidative stress, activations of the NRF2-KEAP1-HO-1 pathway, autophagy, and apoptosis.

Postinfantile Giant Cell Hepatitis in Native and Allograft Livers: A Multi-Institutional Clinicopathologic Study of 70 Cases.

Postinfantile giant cell hepatitis (PIGCH) is a rare hepatitis pattern in adults with variable etiologies and clinical outcomes. We conducted a multi-institutional retrospective study to define the clinicopathologic characteristics of patients with PIGCH. A total of 70 PIGCH cases were identified and reviewed for pathological features, including fibrosis, cholestasis, inflammation, steatosis, necrosis, and apoptosis, as well as the distribution of giant cells and the maximum number of giant cells per high-power field. Demographic and clinical data, including age, sex, laboratory results, etiologies, and follow-up results, were recorded. Among the 70 cases, 40% (28/70) were associated with autoimmune liver diseases, followed by 9 (13%) with unknown etiology, 8 (11%) with viral infection, 5 (7%) with medications, 5 with combined etiologies, and 4 (6%) with malignancies (mostly chronic lymphocytic leukemia). Notably, another 16% were de novo PIGCH in liver allografts, most of which occurred after a rejection event. During follow-up, 26 (37%) patients died of the disease and 44 (63%) were alive. Deceased patients were characterized by older age (mean age, 54.9 vs 45.5 years; P = .02), higher alkaline phosphatase level (mean value, 253.3U/L vs 166.3 U/L; P = .03), higher fibrosis stage (stage 3-4 vs stage 0-2, 57.7% vs 29.6%; P = .03), being more likely to have de novo PIGCH after transplantation (23.1% vs 11.4%; P = .04), and being less likely to have primary autoimmune liver disease etiology (26.9% vs 47.7%; P = .04). These results indicate that PIGCH is a rare pattern of liver injury associated with different etiologies and variable clinical outcomes. Autoimmune liver disease with PIGCH is associated with better survival, whereas de novo PIGCH in allografts is associated with poorer survival. Older age, higher alkaline phosphatase level, and advanced fibrosis are adverse prognostic factors.

Intraductal neoplasms of the pancreatobiliary tract: navigating the alphabet.

Cancers of the pancreatobiliary tract are diseases with unfavourable prognoses. In the last couple of decades, two types of lesions have been described as precursors that precede pancreatobiliary cancers. These include incidental microscopic (flat) lesions known as pancreatic intra-epithelial neoplasia and biliary intra-epithelial neoplasia, and grossly visible, mass-forming lesions (tumoral intra-epithelial neoplasia) including intraductal papillary mucinous neoplasms, intraductal oncocytic papillary neoplasms, intraductal tubulopapillary neoplasms, intraductal papillary neoplasms of the bile duct and intracholecystic papillary neoplasms. Early detection and adequate treatment of these precursor lesions, especially the second group, have the potential to prevent pancreatobiliary cancer or at least improve its prognosis. In this review, we discuss their histopathology and recent updates on molecular profiling of these intraductal neoplasms of the pancreatobiliary tract.

The ovarian-related effects of polystyrene nanoplastics on human ovarian granulosa cells and female mice.

Nanoplastics (NPs) have recently emerged in the context of global plastic pollution. They may be more toxic than macroplastics litter and microplastic fragments due to its abundances, tiny sizes, and cellular accessibility. The female reproductive toxicity of NPs has been widely documented for aquatic animals, but their effects and underlying mechanisms remain poorly understood in mammals. This study aimed to explore the effects of NPs on female reproduction using human ovarian granulosa cells (GCs) and female mice. The accumulation of polystyrene NPs (PS-NPs) in human granulosa-like tumor cells (KGN cells) and the ovaries of female Balb/c mice were evaluated by exposure to fluorescent PS-NPs. Proliferation and apoptosis, reactive oxygen species (ROS), and Hippo signaling pathway-related factors were analyzed in KGN cells. In addition, fertility rate, litter size, ovarian weight and microstructure, follicle development, serum level of anti-Mullerian hormone, and apoptosis in ovaries were examined in female mice. Here, the PS-NPs can penetrate the KGN cells and accumulate in the ovaries. In vitro, 100 µg/ml PS-NPs inhibited proliferation, induced apoptosis, accumulated ROS, activated three key regulators of the Hippo signaling pathway (MST1, LATS1, and YAP1), and downregulated the mRNA levels of CTGF and Cyr61 in KGN cells. Furthermore, salidroside, an antioxidative compound extracted from Rhodiola rosea, alleviated the damage of PS-NPs to KGN and inhibited the activation of the Hippo signal pathway. In vivo, exposure to 1 mg/day PS-NPs resulted in decreased fertility, abnormal ovarian function, and increased ovarian apoptosis in female mice. Overall, our data suggest that PS-NPs cause granulosa cell apoptosis and affect ovarian functions, leading to reduced fertility in female mice, by inducing oxidative stress and dysregulating the Hippo pathway.

Gut Microbiota Dysbiosis Correlates With Long COVID-19 at One-Year After Discharge.

BACKGROUND: Long coronavirus disease 2019 (COVID-19) in recovered patients (RPs) is gradually recognized by more people. However, how long it will last and the underlining mechanism remains unclear. METHODS: We conducted a prospective follow-up study to evaluate the long-term symptoms and clinical indices of RPs at one-year after discharge from Union Hospital, Wuhan, China between December 2020 to May 2021. We also performed the 16S rRNA sequencing of stool samples from RPs and healthy controls (HCs) and analyzed the correlation between the gut microbiota and long COVID-19. RESULTS: In total, 187 RPs were enrolled, among them, 84 (44.9%) RPs reported long COVID-19 symptoms at one-year after discharge. The most common long-term symptoms were cardiopulmonary symptoms, including chest tightness after activity (39/187, 20.9%), palpitations on exercise (27/187, 14.4%), sputum (21/187, 11.2%), cough (15/187, 8.0%) and chest pain (13/187, 7.0%), followed by systemic symptoms including fatigue (34/187, 18.2%) and myalgia (20/187, 10.7%), and digestive symptoms including constipation (14/187, 7.5%), anorexia (13/187, 7.0%), and diarrhea (8/187, 4.3%). Sixty-six (35.9%) RPs presented either anxiety or depression (42/187 [22.8%] and 53/187 [28.8%] respectively), and the proportion of anxiety or depression in the long symptomatic group was significantly higher than that in the asymptomatic group (41/187 [50.6%] vs. 25/187 [24.3%]). Compared with the asymptomatic group, scores of all nine 36-Item Short Form General Health Survey domains were lower in the symptomatic group (all P < 0.05). One hundred thirty RPs and 32 HCs (non-severe acute respiratory syndrome coronavirus 2 infected subjects) performed fecal sample sequencing. Compared with HCs, symptomatic RPs had obvious gut microbiota dysbiosis including significantly reduced bacterial diversities and lower relative abundance of short-chain fatty acids (SCFAs)-producing salutary symbionts such as Eubacterium_hallii_group, Subdoligranulum, Ruminococcus, Dorea, Coprococcus, and Eubacterium_ventriosum_group. Meanwhile, the relative abundance of Eubacterium_hallii_group, Subdoligranulum, and Ruminococcus showed decreasing tendencies between HCs, the asymptomatic group, and the symptomatic group. CONCLUSION: This study demonstrated the presence of long COVID-19 which correlates with gut microbiota dysbiosis in RPs at one-year after discharge, indicating gut microbiota may play an important role in long COVID-19.

Toxic effects of AZD1208 on mouse oocytes and its possible mechanisms.

AZD1208, a pan-inhibitor that can effectively inhibit PIM kinase, is used for the treatment of advanced solid tumors and malignant lymphomas. Numerous studies have proved its curative effects while its potential cellular toxicity on reproduction was still little known. In this study, we investigated the toxic effects of AZD1208 on mouse oocytes. The results showed that AZD1208 treatment did not affect meiotic resumption, but postponed oocyte maturation as indicated by delayed first polar body extrusion. Further mechanistic study showed that AZD1208 treatment delayed spindle assembly. In addition, we found that oocytes treated with AZD1208 showed mitochondrial dysfunction. Abnormal mitochondrial clusters with decreased mitochondrial membrane potential were observed in oocytes during incubation in vitro. Moreover, increased oxidative stress was observed by testing the level of reactive oxygen species. In summary, our results suggest that AZD1208 treatment influences oocyte meiotic progression by causing mitochondrial dysfunctions and subsequent delayed spindle assembly.

Miro1 regulates mitochondrial homeostasis and meiotic resumption of mouse oocyte.

Miro1, a mitochondrial Rho GTPase1, is a kind of mitochondrial outer membrane protein involved in the regulation of mitochondrial anterograde transport and its subcellular distribution. Mitochondria influence reproductive processes of mammals in some aspects. Mitochondria are important for oocyte maturation, fertilization and embryonic development. The purpose of this study was to evaluate whether Miro1 regulates mouse oocyte maturation by altering mitochondrial homeostasis. We showed that Miro1 was expressed in mouse oocyte at different maturation stages. Miro1 mainly distributed in the cytoplasm and around the spindle during oocyte maturation. Small interference RNA-mediated Miro1 depletion caused significantly abnormal distribution of mitochondria and endoplasmic reticulum as well as mitochondrial dysfunction, resulting in severely impaired germinal vesicle breakdown (GVBD) of mouse oocytes. For those oocytes which went through GVBD in the Miro1-depleted group, part of them were inhibited in meiotic prophase I stage with abnormal chromosome arrangement and scattered spindle length. Our results suggest that Miro1 is essential for maintaining the maturation potential of mouse oocyte.

Donor-derived duodenal adenocarcinoma of a bladder-drained pancreas allograft.

The subset of the population that received bladder-drained allograft pancreata during peak utilization of the technique in the 1990s is approaching 20-30 postoperative years. This time frame is salient, as it parallels the time in which patients in the urologic literature develop adenocarcinomas after bladder reconstruction using gastrointestinal segments. We present the case of a 57-year-old simultaneous pancreas/kidney recipient who presented with microhematuria twenty-four years after transplantation and was found to have an adenocarcinoma of the duodenum of his failed, bladder-drained pancreas. After allograft pancreatectomy/duodenectomy, he remains disease-free eleven months postoperatively. As this patient population ages, practitioners should consider pathology of the donor duodenum and pancreas in recipients who present with gross or microscopic hematuria.

Infiltration pattern predicts metastasis and progression better than the T-stage and grade in pancreatic neuroendocrine tumors: a proposal for a novel infiltration-based morphologic grading.

The advancing edge profile is a powerful determinant of tumor behavior in many organs. In this study, a grading system assessing the tumor-host interface was developed and tested in 181 pancreatic neuroendocrine tumors (PanNETs), 63 of which were <=2 cm. Three tumor slides representative of the spectrum (least, medium, and most) of invasiveness at the advancing edge of the tumor were selected, and then each slide was scored as follows. Well-demarcated/encapsulated, 1 point; Mildly irregular borders and/or minimal infiltration into adjacent tissue, 2 points; Infiltrative edges with several clusters beyond the main tumor but still relatively close, and/or satellite demarcated nodules, 3 points; No demarcation, several cellular clusters away from the tumor, 4 points; Exuberantly infiltrative pattern, scirrhous growth, dissecting the normal parenchymal elements, 5 points. The sum of the rankings on the three slides was obtained. Cases with scores of 3-6 were defined as "non/minimally infiltrative" (NI; n = 77), 7-9 as "moderately infiltrative" (MI; n = 68), and 10-15 as "highly infiltrative" (HI; n = 36). In addition to showing a statistically significant correlation with all the established signs of aggressiveness (grade, size, T-stage), this grading system was found to be the most significant predictor of adverse outcomes (metastasis, progression, and death) on multivariate analysis, more strongly than T-stage, while Ki-67 index did not stand the multivariate test. As importantly, cases <=2 cm were also stratified by this grading system rendering it applicable also to this group that is currently placed in "watchful waiting" protocols. In conclusion, the proposed grading system has a strong, independent prognostic value and therefore should be considered for integration into routine pathology practice after being evaluated in validation studies with larger series.

Simultaneous Examination of Eosinophil Infiltration in Esophageal Mucosa and Muscle in Patients with Achalasia: Direct Biopsy of the Esophageal Muscle at Per-oral Endoscopic Myotomy.

BACKGROUND: The relationship between eosinophilic esophagitis (EoE) and achalasia is not completely understood. There have been reports of eosinophilic infiltration of all esophageal layers in patients with achalasia. However, a routine endoscopic biopsy of the muscular layer is usually not feasible. We evaluate the safety and efficacy of muscle layer biopsy during per-oral endoscopic myotomy (POEM) as well as the prevalence of eosinophilic infiltration of the esophageal mucosa and muscular layer in patients with achalasia. PATIENTS AND METHODS: All enrolled patients had diagnosed achalasia and had simultaneous biopsies of the muscular layer at the middle esophagus and distal esophageal sphincter as well as the mucosal layer of the proximal and distal esophagus during POEM. All POEM procedures took place from August 2018 to December 2018 or September 2019 to November 2019. Various demographic, disease-related, and procedure-related data were collected from chart review. Eosinophilic infiltration in the biopsy specimen was examined. KEY RESULTS: Twenty consecutive patients (65% female, age range: 21-84) with a pre-procedure Eckardt score of >6 were enrolled during the study period, with the duration of their achalasia ranging from 1 to 32 years. Eighteen patients had clinical symptomatic improvement after POEM, as defined by an Eckardt score <3. Endoscopic examination did not reveal any signs of eosinophilic esophagitis. Pathologic examination of biopsies revealed eosinophilic infiltration in three of 20 patients (15%) in the distal esophageal mucosa (all <15 eosinophils/HPF) and none in the proximal esophageal mucosa. There was no eosinophilic infiltration in the distal esophageal sphincter and the middle esophageal muscle. No complication was noted due to muscle biopsy. CONCLUSIONS AND INFERENCES: Submucosal tunneling during POEM provides a safe access for direct esophageal muscle biopsy. This is the first report of the simultaneous biopsy of the esophageal mucosa and muscle in patients with achalasia. Contrary to all previously published studies, the association of esophageal eosinophilic infiltration and achalasia was not observed in this small sample study. Based on our findings, immune or autoimmune reaction rather than direct eosinophilic infiltration in the muscle is more likely the cause of achalasia.

Study on the new dynamics and driving factors of soil pH in the red soil, hilly region of South China.

Soil acidification has always been a substantial eco-environmental problem restricting agricultural development in the red soil region of southern China. It is necessary to determine the dynamic change in soil pH in this area to formulate regional agricultural and environmental management measures. Yujiang County, a typical county with red soil acidification in southern China, was selected as the study area. Based on soil data from 1982, 2007, and 2018, the spatiotemporal variation characteristics and the latest changes in soil pH in the county were analyzed. The results show that the soil pH in Yujiang County decreased from 5.66 to 4.74 and then increased to 4.96 from 1982 to 2018, showing a trend of first decreasing and then increasing. According to the spatial distribution characteristics of soil pH, the low soil pH values in the three periods were mainly distributed in the northern mountainous areas with more forestland and dry land area and some southern hilly areas, while the paddy soil pH values in the middle low hilly areas were relatively higher. The soil pH decreased rapidly from 1982 to 2007, showing a large area of acidification. In 2007, the proportions of acidic (4.5 < pH < 5.5) and strongly acidic (pH < 4.5) soils increased by 67.37% and 10.6%, respectively, compared with that in 1982. However, from 2007 to 2018, the soil pH of the whole county increased, and the acidification trend was alleviated, which is of great significance to the regional red soil ecological environment. Through the analysis of the main factors affecting the change in soil pH, it was found that the sharp decline in soil pH in Yujiang County during 1982-2007 was mainly caused by acid rain and excessive nitrogen application. From 2007 to 2018, no significant reduction in nitrogen fertilizer in this area occurred, and although the increase in soil organic matter contributed to alleviating soil acidification, the analysis showed that the decrease in acid rain was the main reason for the rise in soil pH in Yujiang County. At the same time, notably, there is a large area of soil in the area that is still acidic, and effective control of soil acidification is still an important ecological and environmental issue in this area. In order to further improve the pH value of soil in red soil region, it is suggested that on the basis of continuous improvement of acid rain, in addition to increasing soil organic matter by returning straw to field and other measures, appropriate amount of lime or alkaline biochar can be applied to better improve the soil ecological environment in red soil hilly region.

Frequency and clinicopathologic associations of DNA mismatch repair protein deficiency in ampullary carcinoma: Routine testing is indicated.

BACKGROUND: The significance of DNA mismatch repair (MMR) deficiency in ampullary cancers (ACs) has not been established. METHODS: In total, 127 ACs with invasive carcinomas measuring ≥3 mmthat had adequate tissue were analyzed immunohistochemically. RESULTS: MMR loss was detected in 18% of ACs (higher than in colorectal cancers). Twelve tumors with MLH1-PMS2 loss were negative for BRAF V600E mutation, suggesting a Lynch syndrome association. MMR-deficient tumors (n = 23), comparedwith MMR-intact tumors (n = 104), showed a striking male predominance (male:female ratio, 4.7). Although the deficient tumors had slightly larger invasion size (2.7 vs 2.1 cm), they also had more expansile growth and less invasiveness, including less perineural invasion, and they ultimately had lower tumor (T) classification and less lymph node metastasis (30% vs 53%; P = .04). More important, patients who had MMR-deficient tumors had better clinical outcomes, with a 5-year overall survival rate of 68% versus 45% (P = .03), which was even more pronounced in those who had higher Tclassification (5-year overall survival, 69% vs 34%; P = .04). MMR deficiencyhad a statistically significant association with medullary phenotype, pushing-border invasion, and tumor-infiltrating immune cells, and it occurred more frequently in ampullary-duodenal type tumors. Programed cell death-ligand 1 (PD-L1) levels analyzed in the 22 MMR-deficient ACs revealed that all medullary carcinomas were positive. Nonmedullary MMR-deficient carcinomas expressed PD-L1 in 33% of tumors cells according to the criteria for a combined positive score ≥1, but all were negative according to the tumor proportion score≥1 method. CONCLUSIONS: In ACs, MMR deficiency is even more frequent (18%) than in colon cancer and often has a Lynch-suggestive profile, thus routine testing is warranted. Male gender, pushing-border infiltration, ampullary-duodenal origin, medullary histology, and tumor-related inflammation have a significantly higher association with MMR deficiency. MMR-deficient tumors have less aggressive behavior. PD-L1 expression is common in medullary-phenotype ACs, thus immunotherapy should be considered at least for this group.