Esophageal carcinoma with SMARCA4 mutation: a narrative review for this rare entity.

Background and Objective: Esophageal carcinoma with switch/sucrose nonfermenting (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) mutation is a rare variant of malignant esophageal epithelial neoplasm, which is characterized by the loss of SMARCA4/BRG1 protein on immunohistochemistry or alterations in the SMARCA4 gene on sequencing. Only a few case series and case reports of esophageal carcinoma with SMARCA4 mutations have been published in the English literature; the rarity of the disease poses significant diagnostic challenges for surgical pathologists and could potentially lead to delayed or suboptimal patient care. Herein, we reviewed the available literature on esophageal carcinoma with SMARCA4 mutations to discuss its epidemiology, clinical presentation, pathological and molecular features, diagnostic challenges, treatment, and prognosis. Methods: The PubMed, Scopus, Ovid, and Google Scholar databases were extensively reviewed. The references included in the articles were cross-examined to identify any missing articles. We searched for all published literature on esophageal carcinoma with SMARCA4 mutations from inception of the databases to date. Key Content and Findings: Esophageal carcinoma with SMARCA4 mutations is most common in middle-aged and older men. Barrett esophagus and gastroesophageal reflux disease (GERD) are the most associated risk factors. Dysphagia was the most common initial clinical presentation. Esophagogastroduodenoscopy (EGD) is the preferred diagnostic modality. Microscopically, the tumor cells exhibited epithelioid features mixed with variable components of rhabdoid and glandular differentiation. The tumor cells showed variable immunoreactivity for cytokeratin and sometimes weakly expressed neuroendocrine or B-lymphocyte markers (Pax5), which are potential diagnostic pitfalls. Melanoma marker tests showed negative results. The SMARCB1/INI1 protein remains intact, and a definitive diagnosis necessitates the presence of either SMARCA4/BRG1 protein loss or SMARCA4 gene mutations. Esophageal carcinoma with SMARCA4 mutations shows overly aggressive behavior and presents with advanced stages of disease; most patients succumb to the disease within 1 year of initial diagnosis. Conclusions: Esophageal carcinoma with SMARCA4 mutation is an overly aggressive disease, and further research on the affected molecular pathway may help improve its prognosis.

A critical assessment of using ChatGPT for extracting structured data from clinical notes.

Existing natural language processing (NLP) methods to convert free-text clinical notes into structured data often require problem-specific annotations and model training. This study aims to evaluate ChatGPT's capacity to extract information from free-text medical notes efficiently and comprehensively. We developed a large language model (LLM)-based workflow, utilizing systems engineering methodology and spiral "prompt engineering" process, leveraging OpenAI's API for batch querying ChatGPT. We evaluated the effectiveness of this method using a dataset of more than 1000 lung cancer pathology reports and a dataset of 191 pediatric osteosarcoma pathology reports, comparing the ChatGPT-3.5 (gpt-3.5-turbo-16k) outputs with expert-curated structured data. ChatGPT-3.5 demonstrated the ability to extract pathological classifications with an overall accuracy of 89%, in lung cancer dataset, outperforming the performance of two traditional NLP methods. The performance is influenced by the design of the instructive prompt. Our case analysis shows that most misclassifications were due to the lack of highly specialized pathology terminology, and erroneous interpretation of TNM staging rules. Reproducibility shows the relatively stable performance of ChatGPT-3.5 over time. In pediatric osteosarcoma dataset, ChatGPT-3.5 accurately classified both grades and margin status with accuracy of 98.6% and 100% respectively. Our study shows the feasibility of using ChatGPT to process large volumes of clinical notes for structured information extraction without requiring extensive task-specific human annotation and model training. The results underscore the potential role of LLMs in transforming unstructured healthcare data into structured formats, thereby supporting research and aiding clinical decision-making.

INSM1 is a useful neuroendocrine marker to differentiate pancreatic serous cystadenoma from pancreatic well-differentiated neuroendocrine tumors in cytology and surgical specimens.

INTRODUCTION: Differentiating pancreatic serous cystadenoma (SCA) from well-differentiated neuroendocrine tumors (WDNETs) based on histomorphology is critical yet challenging, particularly in small biopsy samples. Our study aimed to examine the expression profile of INSM1 in cytologic and surgical resection specimens from pancreatic SCA to evaluate its potential as a discriminative marker against pancreatic WDNET. METHODS: We characterized INSM1 immunohistochemistry in 34 patients with pancreatic SCA, comprising 23 surgical resections and 11 cytology specimens. As a control, we used 28 cytology specimens from pancreatic WDNET. Clinical information was retrieved through a review of electronic medical records. RESULTS: All 11 pancreatic SCA cytology specimens and 15 of 23 pancreatic SCA surgical resections exhibited absent INSM1 immunostaining. Each of the remaining eight surgical resection specimens demonstrated 1 % immunoreactivity. In contrast, 27 out of 28 (96 %) pancreatic WDNET cytology specimens were positive for INSM1 immunostaining, with a median immunoreactivity of 90 % and a range of 30-90 %. Overall, INSM1 immunostains perform similarly to chromogranin and synaptophysin in pancreatic SCA. CONCLUSIONS: The results indicate that INSM1 immunohistochemistry staining may serve as a useful neuroendocrine marker to differentiate pancreatic SCA from pancreatic WDNET in clinical practice. To our knowledge, this represents the first large-scale study to evaluate INSM1 immunostaining in surgical and cytology specimens from pancreatic SCA.

Deep learning of cell spatial organizations identifies clinically relevant insights in tissue images.

Recent advancements in tissue imaging techniques have facilitated the visualization and identification of various cell types within physiological and pathological contexts. Despite the emergence of cell-cell interaction studies, there is a lack of methods for evaluating individual spatial interactions. In this study, we introduce Ceograph, a cell spatial organization-based graph convolutional network designed to analyze cell spatial organization (for example,. the cell spatial distribution, morphology, proximity, and interactions) derived from pathology images. Ceograph identifies key cell spatial organization features by accurately predicting their influence on patient clinical outcomes. In patients with oral potentially malignant disorders, our model highlights reduced structural concordance and increased closeness in epithelial substrata as driving features for an elevated risk of malignant transformation. In lung cancer patients, Ceograph detects elongated tumor nuclei and diminished stroma-stroma closeness as biomarkers for insensitivity to EGFR tyrosine kinase inhibitors. With its potential to predict various clinical outcomes, Ceograph offers a deeper understanding of biological processes and supports the development of personalized therapeutic strategies.

A Rare Case of a Pancreatic Intraductal Oncocytic Papillary Neoplasm Associated With Invasive Adenocarcinoma Presenting as a Gastric Mass.

The World Health Organization recently recognized intraductal oncocytic papillary neoplasms of the pancreas (IOPNs) as distinct, pre-malignant pancreatic neoplasms. Due to their unique macroscopic and microscopic features, IOPNs are typically easy to diagnose and yield an indolent prognostic outcome. The diagnosis may be more complicated, and the prognosis may differ if an associated invasive carcinoma is present. Owing to the rarity of this entity, the available data is severely limited. Herein, we report a diagnostically challenging case of an IOPN associated with invasive carcinoma, initially presenting as a gastric mass with distinctive radiological and histopathological features.

Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth.

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple upstream stimuli to orchestrate anabolic and catabolic events that regulate cell growth and metabolism. Hyperactivation of mTORC1 signaling is observed in multiple human diseases; thus, pathways that suppress mTORC1 signaling may help to identify new therapeutic targets. Here, we report that phosphodiesterase 4D (PDE4D) promotes pancreatic cancer tumor growth by increasing mTORC1 signaling. GPCRs paired to Gαs proteins activate adenylyl cyclase, which in turn elevates levels of 3',5'-cyclic adenosine monophosphate (cAMP), whereas PDEs catalyze the hydrolysis of cAMP to 5'-AMP. PDE4D forms a complex with mTORC1 and is required for mTORC1 lysosomal localization and activation. Inhibition of PDE4D and the elevation of cAMP levels block mTORC1 signaling via Raptor phosphorylation. Moreover, pancreatic cancer exhibits an upregulation of PDE4D expression, and high PDE4D levels predict the poor overall survival of patients with pancreatic cancer. Importantly, FDA-approved PDE4 inhibitors repress pancreatic cancer cell tumor growth in vivo by suppressing mTORC1 signaling. Our results identify PDE4D as an important activator of mTORC1 and suggest that targeting PDE4 with FDA-approved inhibitors may be beneficial for the treatment of human diseases with hyperactivated mTORC1 signaling.

Pax8 as a useful adjunct marker to differentiate pancreatic serous cystadenoma from clear cell renal cell carcinoma in both cytologic and surgical specimens.

BACKGROUND: Histomorphological differentiation between pancreatic serous cystadenoma (SCA) and clear cell renal cell carcinoma (RCC) can be challenging. We aimed to study Paired box 8 protein (Pax8) expression profile in cytologic and surgical specimens with pancreatic SCA to assess its utility as a differentiating marker from clear cell RCC. METHODS: We characterized Pax8 immunohistochemistry in 33 patients with pancreatic SCA (23 surgical resections and 10 cytology specimens). Nine cytology specimens from metastatic clear cell RCC involving pancreas were used as control tissue. Electronic medical records were reviewed to retrieve clinical information. RESULTS: All 10 pancreatic SCA cytology specimens, and 16 of 23 pancreatic SCA surgical resections showed absent Pax8 immunostaining, while the remaining 7 surgical resection specimens showed 1%-2% immunoreactivities. Islet and lymphoid cells adjacent to the pancreatic SCA expressed Pax8. In contrast, the proportion of Pax8 immunoreactivity ranged from 50 to 90% (average of 76%) in nine cases of metastatic clear cell RCC involving pancreas. Using a 5% immunoreactivity cutoff, all cases of pancreatic SCA are interpreted as negative for Pax8 immunostains while all cases of metastatic clear cell RCC involving pancreas are interpreted as positive for Pax8 immunostains. CONCLUSIONS: These results suggest that Pax8 immunohistochemistry staining can be a useful adjunct marker to differentiate pancreatic SCA from clear cell RCC in clinical practice. To the best of our knowledge, this is the first large-scale study of Pax8 immunostaining on surgical and cytology specimens with pancreatic SCA.

GATA3 positivity is associated with poor prognosis in patients with oesophageal squamous cell carcinoma.

AIMS: GATA-binding protein 3 (GATA3) is a zinc finger transcription factor with diverse biological functions and is an excellent diagnostic marker for breast and urothelial carcinoma. We aimed to study GATA3 expression in oesophageal squamous cell carcinoma (SCC) and its significance with respect to histological features, clinical parameters and overall survival. METHODS: We characterised GATA3 immunohistochemistry in 40 patients with oesophageal SCC. Electronic medical records were reviewed for clinical and follow-up information, as well as patient survival. RESULTS: Eleven (28%) oesophageal SCC were positive for GATA3. The predominant stain patterns were patchy, with either mild or moderate intensities. Patients with GATA3-positive tumours showed significantly shorter overall survival than those with GATA3-negative tumours (p=0.023, Kaplan-Meier survival analysis with log-rank test). In the multivariate Cox proportional hazards regression model, GATA3 positivity was an independent adverse prognostic factor for overall survival (p=0.019, HR 5.671). Surgery, definitive chemotherapy and/or radiotherapy, and initial clinical stage were confirmed as independent prognostic factors. CONCLUSION: To the best of our knowledge, this is the first study to investigate the incidence of GATA3 positivity in oesophageal SCC and showed GATA3 positivity is associated with poor prognosis in oesophageal SCC.

Characterization of Desmoglein 3 (DSG3) as a Sensitive and Specific Marker for Esophageal Squamous Cell Carcinoma.

Although P40 and P63 are both sensitive and specific for routine esophageal squamous cell carcinoma (SCC) diagnosis, we recently showed that P40 and P63 immunoreactivities were significantly lower in well-differentiated SCC than those in higher grade tumors. Therefore, a novel esophageal SCC marker, ideally performing better in well-differentiated SCC, is still needed. We characterized desmoglein 3 (DSG3) immunohistochemistry in esophageal SCC, esophageal adenocarcinoma, small-cell lung carcinoma, and large B-cell lymphoma, alongside P40 and CK5/6. The World Health Organization classification was used to grade tumors as well-differentiated (WD), moderately differentiated (MD), or poorly differentiated (PD). There were 20 WD, 26 MD, and 17 PD components among 39 esophageal SCC cases. All esophageal SCC components showed significant DSG3 immunoreactivity (mean, 80%; range, 30%-100%), and the proportions of DSG3 immunoreactive cells were higher in the WD and MD components than in the PD components. No esophageal adenocarcinoma cases showed more than 10% DSG3 immunoreactivity with only weak cytoplasmic staining. With a 5% immunoreactivity cutoff, DSG3 positivity was 100% in all 63 SCC components, 18% in adenocarcinoma cases, and 0% in small-cell lung carcinoma or large B-cell lymphoma cases. The overall DSG3 specificity was 94%. To the best of our knowledge, this is the first study to characterize DSG3 as a sensitive and specific marker for esophageal SCC.

The Use of Intraoperative Frozen Sections in Guiding the Extent of Pancreatic Resections for Intraductal Papillary Mucinous Neoplasms: A Single Institution Experience and Review of the Literature.

OBJECTIVES: The utility of frozen section evaluation of the pancreatic parenchymal resection margin(s) in the surgical management of intraductal papillary mucinous neoplasm (IPMN) remains controversial. We investigated the frequency of its use in IPMN resections and its impact on achievement of negative final parenchymal margin(s). METHODS: Sixty-two IPMN resections (11 with invasive carcinoma) performed over a 12-year period were studied. RESULTS: Frozen sections of the parenchymal margin(s) were performed on 44 of the 62 resections (71%), 30 (68%) of which had 10 positive and 22 indefinite margins on frozen section. Additional margin resections were performed in 14 of these 30 cases (47%), boosting the complete resection rate from 14% (2 of 14) on the initial margin(s) to 71% (10 of 14) on the final margin(s) (P = 0.002). Overall, negative final parenchymal margin(s) were achieved more frequently when evaluation of the initial margin(s) by frozen section was performed (35 of 44; 80%) than when it was not (11 of 18; 61%) (P = 0.13). CONCLUSIONS: In the intraoperative management of IPMN, frozen sections are highly reliable for margin evaluation and are useful for guiding the extent of pancreatic resection.

Variations of P40 and P63 Immunostains in Different Grades of Esophageal Squamous Cell Carcinoma: A Potential Diagnostic Pitfall.

The distinction of esophageal squamous cell carcinoma (SCC) from adenocarcinoma (adenoCA) using targeted therapies has become critical for small biopsies. In the United States, esophageal SCC is relatively uncommon compared with AdenoCA, with only few detailed immunohistochemical (IHC) studies on esophageal SCC. We characterized p40 and p63 IHC across various grades of squamous differentiation in esophageal SCC and compared their sensitivities between esophageal SCC and adenoCA. Twenty-eight esophageal SCC and 26 esophageal adenoCA (control group) samples were stained for p40, p63, and CK5/6. All hematoxylin-and-eosin-stained SCC slides were reviewed. Tumors were graded according to the World Health Organization classification: well, moderately, or poorly differentiated (WD, MD, and PD, respectively). Considering morphological heterogeneity, individual differentiation components within the same tumor were scored separately (0% to 100%) according to the proportion of immunoreactive cells and marked as positive (≥5%) or negative (<5%). Among 28 esophageal SCC, 15 had mixed intratumoral differentiation. There were 16 WD, 19 MD, and 14 PD components. P40 immunoreactivity was significantly lower in WD than in MD or PD components (P<0.001), P63 immunoreactivity patterns were similar (P<0.001), while CK5/6 showed no differences (P>0.05). The sensitivities for SCC components were 98% (P40), 100% (P63), and 100% (CK5/6), while those for esophageal AdenoCA were significantly lower: 4% (P40), 4% (P63), and 8% (CK5/6). P40 and P63 were sensitive and specific for routine esophageal SCC diagnosis. However, their immunostaining was significantly lower in WD SCC than in higher grade tumors. IHC results for small biopsy specimens should be interpreted carefully, particularly in WD components.

Noninvasive monitoring of hepatic glutathione depletion through fluorescence imaging and blood testing.

Hepatic glutathione plays a key role in regulating redox potential of the entire body, and its depletion is known to increase susceptibility to oxidative stress involved in many diseases. However, this crucial pathophysiological event can only be detected noninvasively with high-end instrumentation or invasively with surgical biopsy, limiting both preclinical research and clinical prevention of oxidative stress-related diseases. Here, we report that both in vivo fluorescence imaging and blood testing (the first-line detection in the clinics) can be used for noninvasive and consecutive monitoring of hepatic glutathione depletion at high specificity and accuracy with assistance of a body-clearable nanoprobe, of which emission and surface chemistries are selectively activated and transformed by hepatic glutathione in the liver sinusoids. These findings open a new avenue to designing exogenous blood markers that can carry information of local disease through specific nanobiochemical interactions back to the bloodstream for facile and rapid disease detection.

Increased Mast Cell Counts and Degranulation in Microscopic Colitis.

OBJECTIVES: Microscopic colitis (MC) is characterized by chronic diarrhea, normal colonoscopy findings, and mucosal inflammation in colonic biopsies and can be classified as collagenous colitis (CC) or lymphocytic colitis (LC). However, the pathogenesis of MC is largely unknown. In this study, we aimed to study mast cell counts and activation in MC. METHODS: We investigated 64 biopsy samples from the surgical pathology database of Indiana University Health, which met the diagnostic criteria for CC or LC along with 20 control samples collected from 2014 to 2015. The specimens were used for the quantification of mast cells by examining the presence of intracellular and extracellular tryptase by immunohistochemistry. RESULTS: In the lamina propria, the mast cell count was higher in both CC and LC groups than the control (mean highest count, 39/high-power field (HPF) vs. 30/HPF vs. 23/HPF; P < 0.01). Extracellular tryptase was present in 10% of control subjects as compared to 41% of CC (P < 0.01). Extracellular tryptase was present in 10% of control subjects as compared to 41% of CC (P < 0.01). Extracellular tryptase was present in 10% of control subjects as compared to 41% of CC (. CONCLUSIONS: The increased mast cell count and degranulation are identified in MC, suggesting that mast cell activation might be involved in the pathogenesis of MC.

The AAA + ATPase Thorase is neuroprotective against ischemic injury.

Neuronal preconditioning in vitro or in vivo with a stressful but non-lethal stimulus leads to new protein expression that mediates a profound neuroprotection against glutamate excitotoxicity and experimental stroke. The proteins that mediate neuroprotection are relatively unknown and under discovery. Here we find that the expression of the AAA + ATPase Thorase is induced by preconditioning stimulation both in vitro and in vivo. Thorase provides neuroprotection in an ATP-dependent manner against oxygen-glucose deprivation (OGD) neurotoxicity or glutamate N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity in vitro. Knock-down of Thorase prevents the establishment of preconditioning induced neuroprotection against OGD or NMDA neurotoxicity. Transgenic overexpression of Thorase provides neuroprotection in vivo against middle cerebral artery occlusion (MCAO)-induced stroke in mice, while genetic deletion of Thorase results in increased injury in vivo following stroke. These results define Thorase as a neuroprotective protein and understanding Thorase signaling could offer a new therapeutic strategy for the treatment of neurologic disorders.

Mucinous intrahepatic cholangiocarcinoma: a distinct variant.

Mucinous variant of intrahepatic cholangiocarcinoma (iCC) is rare, and its clinicopathological features and prognosis are far less clear. Six patients who had iCCs with more than 50% of mucinous component and 79 conventional iCCs were included in the study. The mean size of mucinous and conventional iCCs was 6.2 and 6.0 cm, respectively. Most patients (83%) with mucinous iCC presented at T3 stage or above compared with 28% of the conventional group (P < .01). Three patients with mucinous iCC (50%) died within 1 year. The average survival time of patients with mucinous iCCs was significantly reduced compared with that of the conventional group (9 months versus 2 years; P < .001). Immunohistochemistry was performed on 6 mucinous and 12 conventional iCCs with matched age, sex, and stage, which revealed positive immunoreactivity in MUC1 (83% versus 58%), MUC2 (33% versus 17%), MUC5AC (100% versus 42%), MUC6 (50% versus 0), CK7 (83% versus 83%), CK20 (0 versus 17%), CDX2 (17% versus 0), p53 (67% versus 67%), Smad4 (67% versus 58%), and EGFR (83% versus 42%) in mucinous and conventional iCCs, respectively. Molecular studies showed one mucinous iCC with KRAS G12C mutation and no BRAF or IDH1/2 mutations. Mucinous iCC is a unique variant that constitutes 7% of iCCs. It is more immunoreactive for MUC1, MUC2, MUC5AC, and MUC6. Unlike adenocarcinomas of colorectal primary, mucinous iCCs are often CK7+/CK20-/CDX2- and microsatellite stable. Patients with mucinous iCC likely present at advanced stage upon diagnosis with shorter survival time compared with the conventional counterparts.

Multivisceral transplant is a viable treatment option for patients with non-resectable intra-abdominal fibromatosis.

BACKGROUND: Intra-abdominal fibromatosis often involves the mesentery root which is non-resectable by conventional surgery. Multivisceral transplant (MVT), as a potential cure to non-resectable fibromatosis, has rarely been reported and the prognosis is unknown. METHODS: Six patients who underwent MVT for intra-abdominal fibromatosis were reviewed. Clinicopathological features, immunohistochemistry for ß-catenin, p53, and Ki67, and outcomes were evaluated. Appropriate data for comparative analysis were obtained from a cohort of 24 patients who underwent conventional resection for intra-abdominal fibromatosis. RESULTS: Among six MVT patients, four had familial adenomatous polyposis (FAP). Two patients had an initial intestinal transplantation, three had multiple prior surgeries, and two had adjuvant therapy. One patient died of hemorrhagic stroke shortly after MVT, and five patients (83%) survived with a median follow-up of 64 months. The 1-year and 5-year survival rates were 67% for all five patients. Two patients had recurrences after MVT and one of them had FAP. In comparison, six of 24 patients who underwent conventional surgery had FAP; six (25%) had recurrences and three had FAP. For FAP patients; the mean recurrence time was 13 months for MVT versus 6 months for conventional surgery. Ki67 proliferative index, ß-catenin, and p53 expression did not significantly correlate to recurrence. CONCLUSIONS: Multivisceral transplant (MVT) is a viable option for patients who have non-resectable intra-abdominal fibromatosis with promising surviving rates, although recurrence still occurs. Surgical margin, Ki67 proliferative index, ß-catenin, and p53 expression are not predicative for recurrence of fibromatosis.

Cytomorphological Features Useful to Prevent Errors in the Diagnosis of Pancreatic Adenocarcinoma by Fine Needle Aspiration Cytology.

OBJECTIVES: Endoscopic ultrasound-guided fine-needle aspiration (FNA) is now widely used as a primary tool to diagnose pancreatic neoplasms. However, criteria that can reduce the risk of overdiagnosing pancreatic adenocarcinoma by FNA have not been adequately defined in the literature. This study aims to identify characteristic cytomorphological features that are helpful in distinguishing pancreatic adenocarcinoma from its mimics. STUDY DESIGN: Five false-positive FNA cases (group A) diagnosed as adenocarcinoma (4 cases) and suspicious for adenocarcinoma (1 case) by FNA, were identified by searching our laboratory information system. Cytomorphological features of group A cases were compared to 12 true-positive, histologically confirmed FNA cases (group B). RESULTS: Subsequent histological follow-ups of 5 misdiagnosed FNA cases showed 2 cases of intraductal papillary mucinous neoplasm with focal high-grade dysplasia, 1 case attributed to tumor contamination from a gastroesophageal junction adenocarcinoma, and 2 cases of pancreatic intraepithelial neoplasia (PanIN1/reactive change and PanIN2, respectively). Cytomorphological features present in both groups A and B included nuclear enlargement/overlapping, mild to moderate anisonucleosis, granular chromatin and prominent nucleoli. However, 1 or more of these 4 characteristic morphological features such as 3-dimensional cluster with cell disorientation, isolated malignant cells, irregular nuclear contour/nuclear grooves/notches (>5% atypical cell population), and marked nuclear size variation 1:4 or higher was mainly present in adenocarcinoma. CONCLUSIONS: A combination of at least 2 of these 4 characteristic cytomorphological features needs to be present before rendering an unequivocal diagnosis of adenocarcinoma. Using these strict cytological criteria would have eliminated these false-positive diagnoses.

High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death.

Neuronal loss caused by ischemic injury, trauma, or disease can lead to devastating consequences for the individual. With the goal of limiting neuronal loss, a number of cell death pathways have been studied, but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators, we performed a high-content genome-wide screening of short, interfering RNA (siRNA) with an siRNA library in murine neural stem cells after exposure to N-methyl-N-nitroso-N'-nitroguanidine (MNNG), which leads to DNA damage and cell death. Eighty genes were identified as key mediators for cell death. Among them, 14 are known cell death mediators and 66 have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways, and/or protein complexes that may participate in cell death. Of the 66 genes, we selected CCR3 for further evaluation and found that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation-induced cell death, and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together, our findings suggest that CCR3 is a previously unknown mediator of cell death. Future identification of the neural cell death network in which CCR3 participates will enhance our understanding of the molecular mechanisms of neural cell death.

Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity.

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

Botch is a γ-glutamyl cyclotransferase that deglycinates and antagonizes Notch.

Botch promotes embryonic neurogenesis by inhibiting the initial S1 furin-like cleavage step of Notch maturation. The biochemical process by which Botch inhibits Notch maturation is not known. Here, we show that Botch has γ-glutamyl cyclotransferase (GGCT) activity that deglycinates Notch, which prevents the S1 furin-like cleavage. Moreover, Notch is monoglycinated on the γ-glutamyl carbon of glutamate 1,669. The deglycinase activity of Botch is required for inhibition of Notch signaling both in vitro and in vivo. When the γ-glutamyl-glycine at position 1,669 of Notch is degylcinated, it is replaced by 5-oxy-proline. These results reveal that Botch regulates Notch signaling through deglycination and identify a posttranslational modification of Notch that plays an important role in neurogenesis.