A case of metastatic lymphoepithelial carcinoma of parotid gland identified on 68gallium DOTA-[Tyr3] octreotate PET CT.

The authors present the case of a 59-year-old lady diagnosed with lymphoepithelial carcinoma (LEC) of the left parotid gland. The primary tumour was identified using contrast-enhanced CT, and diagnosis was confirmed via fine needle aspiration cytology and immunohistochemistry. Staging using fluorine-18 fluorodeoxyglucose PET CT revealed regional nodal metastases, while no distant metastasis was evident. Following radical radiotherapy, a favourable locoregional response was observed on MRI, yet the patient's plasma Epstein-Barr virus load continued to rise. Given her primary tumour's somatostatin receptor type 2 (SSTR2) positivity, gallium-68 DOTA-[Tyr3] octreotate PET CT (68Ga-DOTATATE PET CT) was performed, revealing multiple distant metastases with DOTATATE avidity. Despite attempts at palliative chemotherapy and immunotherapy, disease progression led to the decision for the best supportive care. The unique presentation of metastatic LEC on 68Ga-DOTATATE PET CT suggests a potential role for SSTR2-targeted imaging in diagnosis and management.

MR Spectroscopy Assessment of Daily Variations of GABA Levels within the Parietal Lobe and Anterior Cingulate Gyrus Regions of Healthy Young Adults.

BACKGROUND: The changes that occur in the gamma-aminobutyric acid (GABA) levels within specific brain regions throughout the day are less clear. PURPOSE: To evaluate the daily fluctuations of GABA levels within the parietal lobe (PL) and anterior cingulate gyrus (ACC) regions and explore their association with melatonin (MT) levels, heart rate (HR), and blood pressure. STUDY TYPE: Prospective. SUBJECTS: 26 healthy young adults (15 males and 11 females aged 22-27 years). FIELD STRENGTH/SEQUENCE: 3.0T, T1-weighted imaging, Mescher-Garwood point resolved spectroscopy (MEGA-PRESS) sequence. ASSESSMENT: The acquired GABA signal contained the overlapping signals of macromolecules and homocarnosine, hence expressed as GABA+. The creatine (Cr) signal was applied as an endogenous reference. The GABA+, GABA+/Cr were measured at six different time points (1:00, 5:00, 9:00, 13:00, 17:00, and 21:00 hours) using MEGA-PRESS. The blood pressure, HR and sputum MT levels, were also acquired. STATISTICAL TESTS: The one-way repeated-measures analysis of variance (ANOVA) was used to evaluate the GABA, blood pressure, HR, and MT levels throughout the day. A general linear model was used to find the correlation between GABA and blood pressure, HR, and MT. P < 0.05 was statistically significant. RESULTS: Significant variations in GABA+/Cr and GABA+ levels were observed throughout the day within the PL region. The lowest levels were recorded at 9:00 hour (GABA+/Cr: 0.100 ± 0.003，GABA+:1.877 ± 0.051 i.u) and the highest levels were recorded at 21:00 hour (GABA+/Cr: 0.115 ± 0.003, GABA+:2.122 ± 0.052 i.u). The MT levels were positively correlated with GABA+/Cr (r = 0.301) and GABA+ (r = 0.312) within the ACC region. DATA CONCLUSION: GABA+/Cr and GABA+ in ACC are positively correlated with MT. GABA levels in the PL have diurnal differences. These findings may indicate that the body's GABA level change in response to the light-dark cycle. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 2.

Behavior-relevant top-down cross-modal predictions in mouse neocortex.

Animals adapt to a constantly changing world by predicting their environment and the consequences of their actions. The predictive coding hypothesis proposes that the brain generates predictions and continuously compares them with sensory inputs to guide behavior. However, how the brain reconciles conflicting top-down predictions and bottom-up sensory information remains unclear. To address this question, we simultaneously imaged neuronal populations in the mouse somatosensory barrel cortex and posterior parietal cortex during an auditory-cued texture discrimination task. In mice that had learned the task with fixed tone-texture matching, the presentation of mismatched pairing induced conflicts between tone-based texture predictions and actual texture inputs. When decisions were based on the predicted rather than the actual texture, top-down information flow was dominant and texture representations in both areas were modified, whereas dominant bottom-up information flow led to correct representations and behavioral choice. Our findings provide evidence for hierarchical predictive coding in the mouse neocortex.

Pathogenesis and therapeutic implications of EBV-associated epithelial cancers.

Epstein-Barr virus (EBV), one of the most common human viruses, has been associated with both lymphoid and epithelial cancers. Undifferentiated nasopharyngeal carcinoma (NPC), EBV associated gastric cancer (EBVaGC) and lymphoepithelioma-like carcinoma (LELC) are amongst the few common epithelial cancers that EBV has been associated with. The pathogenesis of EBV-associated NPC has been well described, however, the same cannot be said for primary pulmonary LELC (PPLELC) owing to the rarity of the cancer. In this review, we outline the pathogenesis of EBV-associated NPC and EBVaGCs and their recent advances. By drawing on similarities between NPC and PPLELC, we then also postulated the pathogenesis of PPLELC. A deeper understanding about the pathogenesis of EBV enables us to postulate the pathogenesis of other EBV associated cancers such as PPLELC.

68 Ga-DOTATATE PET/CT of Metastatic Lymphoepithelial Carcinoma of Parotid Gland.

ABSTRACT: We present a case of a 59-year-old woman with lymphoepithelial carcinoma of left parotid gland. She was treated with radical radiotherapy, but her plasma Epstein-Barr virus DNA load continued to increase despite good locoregional response. As her primary tumor was positive for somatostatin receptor type 2, we performed 68 Ga-DOTATATE PET/CT, which revealed multiple DOTATATE-avid distant metastases.

Assessment of neurotransmitter imbalances within the anterior cingulate cortex in women with primary dysmenorrhea: An initial proton magnetic resonance spectroscopy study.

PURPOSE: The neural pathophysiology underlying primary dysmenorrhea (PDM), which leads to poor mode and changes in central pain modulatory systems, remains largely unknown. The objective of this study was to investigate the changes in glutamate/glutamine (Glx) and gamma-aminobutyric acid (GABA+) levels within anterior cingulate cortex (ACC), and their associations with clinical indicators in PDM women. METHODS: Using 3 T proton magnetic resonance spectroscopy (1H-MRS), we acquired and compared ACC-Glx and ACC-GABA+ levels in PDMs (N = 41) and age- and education-matched healthy controls (HCs) (N = 39) during both the menstrual and periovulatory phases, and between menstrual and periovulatory phases within each group. Total creatine (Cr referencing) level was used as an endogenous reference. The correlations of ACC-neurotransmitter levels with clinical characteristics and the correlations of ACC-Glx with ACC-GABA+ levels in the two groups were analyzed. RESULTS: Compared to HCs or the periovulatory phase, PDMs exhibited significantly increased ACC-Glx levels (p < 0.05) during the menstrual phase. Positive correlations between GABA+ and Glx levels (r = 0.385, p = 0.025) were found in PDMs during the menstrual phase. ACC-GABA+ levels were associated with self-rating distress scale (SDS) scores (GABA+/Cr: r = 0.369, p = 0.045) and pain catastrophizing scale (PCS) scores (GABA+/Cr: r = 0.373, p = 0.042) in PDM group in only the menstrual phase. CONCLUSION: Our study represents the first report of ACC-GABA+/Glx imbalances in PDMs during the menstrual phase, which may underlie the mechanisms mediating depression and painful catastrophic symptoms.

The distinct clinical trajectory, metastatic sites, and immunobiology of microsatellite-instability-high cancers.

Microsatellite-instability-high (MSI-H) cancers form a spectrum of solid organ tumors collectively known as Lynch Syndrome cancers, occurring not only in a subset of colorectal, endometrial, small bowel, gastric, pancreatic, and biliary tract cancers but also in prostate, breast, bladder, and thyroid cancers. Patients with Lynch Syndrome harbor germline mutations in mismatch repair genes, with a high degree of genomic instability, leading to somatic hypermutations and, therefore, oncogenesis and cancer progression. MSI-H cancers have unique clinicopathological characteristics compared to their microsatellite-stable (MSS) counterparts, marked by a higher neoantigen load, immune cell infiltration, and a marked clinical response to immune checkpoint blockade. Patients with known Lynch Syndrome may be detected early through surveillance, but some patients present with disseminated metastatic disease. The treatment landscape of MSI-H cancers, especially colorectal cancers, has undergone a paradigm shift and remains to be defined, with immune checkpoint blockade coming to the forefront of treatment strategies in the stage IV setting. We summarize in this review the clinical features of MSI-H cancers with a specific interest in the pattern of spread or recurrence, disease trajectory, and treatment strategies. We also summarize the tumor-immune landscape and genomic profile of MSI-H cancers and potential novel therapeutic strategies.

Elevated GABA level in the precuneus and its association with pain intensity in patients with postherpetic neuralgia: An initial proton magnetic resonance spectroscopy study.

PURPOSE: This study aimed to explore the changes in gamma-aminobutyric acid (GABA) and glutamate (Glu) levels, and their correlations with clinical indicators in patients with postherpetic neuralgia (PHN). METHODS: A totally of 22 PHN patients and 21 sex-, age-, and education-matched healthy controls (HCs) underwent proton magnetic resonance spectroscopy scanning. The spectral data of GABA in the precuneus was obtained by Mescher-Garwood point-resolved spectroscopy, whereas the spectral data of Glu, total N-acetyl-l-aspartic acid (tNAA) and total choline (tCho) were obtained by point-resolved spectroscopy. The pain intensity of PHN was assessed by numeric rating scales (NRS). The edited GABA signal was displayed as GABA+ due to overlapping macromolecules and homocarnosine signals. Total creatine (tCr) level in individual was used as an endogenous reference. The neurometabolites levels of PHN patients were compared with those of healthy individuals and the correlations with clinical variables (pain duration and intensity) were analyzed. RESULTS: PHN patients had higher GABA+/tCr levels in the precuneus than HCs (P = 0.009), with no significant differences in the levels of Glu/ tCr, tNAA/ tCr and tCho/ tCr (all P > 0.05). The GABA+/ tCr levels were positively correlated with the NRS scores of the PHN patients (r = 0.473, P = 0.030). CONCLUSION: Increased GABA+/tCr levels in the precuneus and their association with pain intensity of PHN patients suggested a key role of the abnormalities of regional GABAergic neurons in the pathophysiological mechanisms of pain in PHN.

Pre-treatment spectral CT combined with CT perfusion can predict hemorrhagic transformation after thrombolysis in patients with acute ischemic stroke.

OBJECTIVE: To investigate the value of pre-treatment spectral CT angiography (CTA) in predicting hemorrhagic transformation (HT) after intravenous thrombolysis (IVT) treatment in acute ischemic stroke (AIS) patients. MATERIALS AND METHODS: AIS patients who underwent IVT with recombinant tissue plasminogen activator and pre-treatment head and neck spectral CTA and head CT perfusion (CTP) from January 2018 to June 2020 were reviewed retrospectively. Finally, 20 patients were included in the HT group and 22 age-matched patients were included in the non-HT group. Spectral and CTP parameters of the region of interest on pre-treatment CTA axial raw images and CTP images, including the infarct core (IC) and ischemic penumbral (IP) regions, were recorded. The differences in clinical variables, CTP, collateral scores and spectral parameters between the two groups were analyzed. Three multivariate logistic regression models were then developed, where model 1 included clinical and spectral parameters, model 2 included clinical and CTP parameters and a combined model included clinical, CTP, and spectral parameters. Receiver operating characteristic analysis was used to evaluate the performance of the multivariate model. RESULTS: Patients with HT had higher Safe Implementation of Treatments in Stroke (SITS) score (p = 0.023), the volume of perfusion lesions (p = 0.005), the volume of IP (p = 0.003), the mean transit time (MIT) in the IC area (p = 0.012), as well as the TTP in IP area (p = 0.015) compared with patients without HT. The HT group showed significantly lower CBF in the IC area (p = 0.019), iodine concentration (p = 0.017) and the effective atomic number (p = 0.024) in the IP area than non-HT group. And the slope of the spectral curve of the HT group in the IP region was larger than that of the non-HT group (p = 0.023). Gender, age, SITS score, the volume of entire perfusion lesion, CBF and MIT in the IC area, TTP in the IP area, as well as iodine concentration in the IP area were included in the final multivariate model for predicting HT. And CBF in the IC area (OR = 0.779, 95 % CI:0.609-0.996, p = 0.046) as well as the iodine concentration of IP area (OR = 0.343, 95 % CI: 0.131-0.901, p = 0.030) were proved to be independent predictors for HT. The combined model including clinical, spectral, and CTP parameters, showed improved accuracy compared to the other two models, while the Delong test did not suggest a statistically significant difference (both p values > 0.05). CONCLUSIONS: The iodine concentration of IP area derived from pre-treatment spectral CTA was an independent predictor of HT after IVT treatment for AIS patients. Moreover, multivariate models combined with clinical, spectral, and CTP parameters may be able to predict HT.

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients.

Genome-wide analysis of cell-free DNA methylation profile is a promising approach for sensitive and specific detection of many cancers. However, scaling such assays for clinical translation is impractical because of the high cost of whole-genome bisulfite sequencing. We show that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors most of the cancer-specific methylation signature. Here, we report on the simple and effective heat enrichment of CpG-rich regions for bisulfite sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation and quantitative tracking of colorectal cancer patient's response to treatment at much reduced sequencing cost suitable for frequent monitoring. We also show tumor epigenetic subtyping using Heatrich-BS, which could enable patient stratification. Heatrich-BS holds great potential for highly scalable screening and monitoring of cancer using liquid biopsy.

Resting-State Functional Connectivity Analyses: Brain Functional Reorganization in a Rat Model of Postherpetic Neuralgia.

Postherpetic neuralgia (PHN) is a chronic neuropathic pain syndrome, similar to other chronic pains, the mechanisms of which are not fully understood. To further understand the neural mechanism of this chronic pain and its transition, we performed functional magnetic resonance imaging (fMRI) scans on PHN rat models. Twelve PHN rat models were established by intraperitoneal injection of resiniferatoxin, with an additional 12 rats serving as controls. Nociceptive behavioral tests were performed on these rats and fMRI scans were performed on days 7 and 14 after modeling. Functional connection (FC) analysis was used to investigate the brain FC alterations associated with chronic pain in PHN rats, with the anterior cingulate cortex (ACC) as a seed. Nociceptive behavioral tests showed that PHN rats presented symptoms similar to those of PHN patients. FC analysis showed that compared to the control group, the PHN group showed different FC patterns on days 7 and 14. As can be seen, the brain FC alterations in the rat model of PHN changed dynamically, shifting from brain regions processing sensory information to regions processing emotions and motives.

Altered functional connectivity associated with cognitive impairment in neuromyelitis optica spectrum disorder.

BACKGROUND: Cognitive impairment is one of the common symptoms in patients with neuromyelitis optica spectrum disorder (NMOSD). However, the underlying mechanism remains unclear. Resting-state functional magnetic resonance imaging (rs-fMRI) offers the opportunity to reveal the patterns of brain activity in patients with different cognitive states. Accordingly, this study investigated functional connectivity (FC) abnormalities within and between the main cognitive networks in cognitively impaired (CI) patients with NMOSD and their correlations with cognitive performance. METHODS: Thirty-four patients with NMOSD and 39 healthy controls (HC) were included. Neuropsychological evaluations and rs-fMRI scanning were performed. Patients were classified as CI (n = 16) or cognitively preserved (CP; n = 18) according to neuropsychological evaluations. Seven components representing six main cognitive networks were selected by group independent component analysis. The differences in inter- and intranetwork FC among CI, CP, and HC groups were assessed. The correlation between FC values and neuropsychological data in NMOSD was calculated. RESULTS: The CI group showed decreased intranetwork connectivity in the posterior default mode network (pDMN) compared with the HC group (P < 0.05, GRF corrected), and decreased internetwork connectivity between the salience network (SN) and pDMN, and between the SN and right frontoparietal network (rFPN) compared with CP and HC groups. The altered FC values were significantly correlated with cognitive performance in the whole NMOSD group. CONCLUSION: The disconnection within the pDMN and between the SN and pDMN or rFPN might suggest the neural substrates underlying cognitive impairment in NMOSD.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer.

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Impact of cancer diagnoses on the outcomes of patients with COVID-19: a systematic review and meta-analysis.

BACKGROUND: The COVID-19 has caused significant mortality and morbidity across the globe. Patients with cancer are especially vulnerable given their immunocompromised state. We aimed to determine the proportion of COVID-19 patients with cancer, their severity and mortality outcomes through a systematic review and meta-analysis (MA). METHODS: Systematic review was performed through online databases, PubMed, Medline and Google Scholar, with keywords listed in the Methods section (1 November 2019-31 December 2020). Studies with clinical outcomes of at least 10 COVID-19 patients and at least one with a diagnosis of cancer were included. The studies for MA were assessed with PRISMA guidelines and appraised with Newcastle-Ottawa Scale. The data were pooled using a random-effects model using STATA software. The main outcomes were planned before data collection, including proportion of patients with cancer among COVID-19 populations, relative risk (RR) of severe outcomes and death of patients with cancer compared with general COVID-19 patients. RESULTS: We identified 57 case series (63 413 patients), with 230 patients with cancer with individual patient data (IPD). We found that the pooled proportion of cancer among COVID-19 patients was 0.04 (95% CI 0.03 to 0.05, I2=97.69%, p<0.001). The pooled RR of death was 1.44 (95% CI 1.19 to 1.76) between patients with cancer and the general population with COVID-19 infection. The pooled RR of severe outcome was 1.49 (95% CI 1.18 to 1.87) between cancer and general COVID-19 patients. The presence of lung cancer and stage IV cancer did not result in significantly increased RR of severe outcome. Among the available IPD, only age and gender were associated with severe outcomes. CONCLUSION: Patients with cancer were at a higher risk of severe and death outcomes from COVID-19 infection as compared with general COVID-19 populations. Limitations of this study include publication bias. A collaborative effort is required for a more complete database.

Decreased Brain GABA Levels in Patients with Migraine Without Aura: An Exploratory Proton Magnetic Resonance Spectroscopy Study.

Increasing neurophysiological studies had revealed that regional excitation-inhibition imbalance in the brain played a key role in the pathogenesis of migraine. This study aimed to explore the alterations in gamma-aminobutyric acid (GABA) and glutamate/glutamine complex (Glx) levels in the anterior cingulate gyrus (ACC) and medial prefrontal lobe (mPFC) of patients with migraine without aura (MWoA) and investigate the correlation between neurotransmitter levels and clinical indicators. A total of 28 patients with MWoA and 28 sex-, age-, and education level-matched healthy controls (HCs) underwent single-voxel proton magnetic resonance spectroscopy scanning at 3.0 Tesla. MEscher-Garwood Point RESolved Spectroscopy (MEGA-PRESS) sequence was performed to acquire the spectral data of GABA and Glx in the ACC and mPFC. The clinical indicators and anxiety-depression states of all participants were assessed. The acquired GABA signal contained the overlapping signals of macromolecules and homocarnosine, hence expressed as GABA+. The creatine (Cr) signal was applied as an endogenous reference. We observed that GABA+/Cr levels were significantly lower in ACC and mPFC of patients with MWoA than of HCs, with no significant difference in Glx levels. Negative correlations between GABA+/Cr levels and attack frequency were found in the ACC and mPFC regions of patients. These results suggested that there might be a close relationship between ACC and mPFC GABAergic neurons abnormalities and the pathophysiological mechanisms of MWoA. It might be beneficial to targeted treatment for patients with MWoA.

Reduced GABA levels in the medial prefrontal cortex are associated with cognitive impairment in patients with NMOSD.

BACKGROUND: Cognitive impairment is a symptom present in part of patients with neuromyelitis optica spectrum disorder (NMOSD) and its pathophysiology is unknown. Dysfunction of the GABAergic/glutamatergic pathways involving inhibitory and excitatory neurotransmitters have been implicated in several neurological disorders. This study aimed to investigate the changes in inhibitory gamma-aminobutyric acid (GABA) and excitatory glutamate and glutamine (Glx) neurotransmitter levels and their correlations with cognitive functions in patients with NMOSD. METHODS: A total of 29 patients with NMOSD and 28 sex-, age-, and education-matched healthy controls (HCs) were included in the study. All participants underwent clinical and cognitive assessments and proton magnetic resonance spectroscopy scanning. Meshcher-Garwood point-resolved spectroscopy was used to measure GABA and Glx levels in the medial prefrontal cortex (mPFC) and left thalamus. Total creatine (tCr) was applied as an internal reference. The GABA and Glx levels in the patient group were compared with those in HCs and correlated with cognitive scores and clinical variables. RESULTS: Patients with NMOSD showed lower GABA+/tCr levels in the mPFC compared with HCs (P = 0.028). The GABA+/tCr levels in the mPFC were significantly associated with verbal memory performance (r = 0.462, P = 0.027) and overall cognition (r = 0.440, P = 0.035) in the NMOSD group. The GABA+/tCr levels in the left thalamus or Glx/tCr levels in both regions were not significantly different between groups, nor were they related to any cognitive domain in patients with NMOSD (all P values > 0.05). CONCLUSION: The GABA+ levels in the mPFC decreased and correlated with cognitive dysfunction in patients with NMOSD, suggesting that the changes in regional GABA+ levels might be a potential metabolic feature of cognitive decline in patients with NMOSD.

Epstein-Barr Virus Epithelial Cancers-A Comprehensive Understanding to Drive Novel Therapies.

Epstein-Barr virus (EBV) is a ubiquitous oncovirus associated with specific epithelial and lymphoid cancers. Among the epithelial cancers, nasopharyngeal carcinoma (NPC), lymphoepithelioma-like carcinoma (LELC), and EBV-associated gastric cancers (EBVaGC) are the most common. The role of EBV in the pathogenesis of NPC and in the modulation of its tumour immune microenvironment (TIME) has been increasingly well described. Much less is known about the pathogenesis and tumour-microenvironment interactions in other EBV-associated epithelial cancers. Despite the expression of EBV-related viral oncoproteins and a generally immune-inflamed cancer subtype, EBV-associated epithelial cancers have limited systemic therapeutic options beyond conventional chemotherapy. Immune checkpoint inhibitors are effective only in a minority of these patients and even less efficacious with molecular targeting drugs. Here, we examine the key similarities and differences of NPC, LELC, and EBVaGC and comprehensively describe the clinical, pathological, and molecular characteristics of these cancers. A deeper comparative understanding of these EBV-driven cancers can potentially uncover targets in the tumour, TIME, and stroma, which may guide future drug development and cast light on resistance to immunotherapy.

Identification of Pattern Completion Neurons in Neuronal Ensembles Using Probabilistic Graphical Models.

Neuronal ensembles are groups of neurons with coordinated activity that could represent sensory, motor, or cognitive states. The study of how neuronal ensembles are built, recalled, and involved in the guiding of complex behaviors has been limited by the lack of experimental and analytical tools to reliably identify and manipulate neurons that have the ability to activate entire ensembles. Such pattern completion neurons have also been proposed as key elements of artificial and biological neural networks. Indeed, the relevance of pattern completion neurons is highlighted by growing evidence that targeting them can activate neuronal ensembles and trigger behavior. As a method to reliably detect pattern completion neurons, we use conditional random fields (CRFs), a type of probabilistic graphical model. We apply CRFs to identify pattern completion neurons in ensembles in experiments using in vivo two-photon calcium imaging from primary visual cortex of male mice and confirm the CRFs predictions with two-photon optogenetics. To test the broader applicability of CRFs we also analyze publicly available calcium imaging data (Allen Institute Brain Observatory dataset) and demonstrate that CRFs can reliably identify neurons that predict specific features of visual stimuli. Finally, to explore the scalability of CRFs we apply them to in silico network simulations and show that CRFs-identified pattern completion neurons have increased functional connectivity. These results demonstrate the potential of CRFs to characterize and selectively manipulate neural circuits.SIGNIFICANCE STATEMENT We describe a graph theory method to identify and optically manipulate neurons with pattern completion capability in mouse cortical circuits. Using calcium imaging and two-photon optogenetics in vivo we confirm that key neurons identified by this method can recall entire neuronal ensembles. This method could be broadly applied to manipulate neuronal ensemble activity to trigger behavior or for therapeutic applications in brain prostheses.

Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition.

As glutamine plays a central role in cancer metabolism, inhibition of glutaminolysis has become an ideal anticancer therapeutic target. However, glutaminolysis inhibition leads to activation of autophagy, which compromises its antitumor effect. Hence, we investigated the mechanism underlying glutaminolysis inhibition-induced pro-survival autophagy. Methods: High-throughput sequencing was performed on colorectal cancer (CRC) cells before and after glutaminolysis inhibition to identify differentially expressed genes. Activating transcription factor 4 (ATF4) pathway enrichment in glutaminolysis inhibited cells was identified through gene set enrichment analysis. ATF4 expression was assessed by quantitative real-time PCR (qRT-PCR) and western blotting. The function of ATF4 on mechanistic target of rapamycin (mTOR) regulation was assessed by western blotting. Luciferase reporter assays and chromatin immunoprecipitation were used to confirm the regulation of DNA damage inducible transcript 4 (DDIT4) by ATF4. mRNA half-life assays, RNA immunoprecipitation, qRT-PCR and western blotting were performed to determine the relationship between FTO alpha-ketoglutarate dependent dioxygenase (FTO), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), and ATF4. ATF4 regulation of pro-survival autophagy was measured by tandem monomeric red fluorescent protein-green fluorescent protein fluorescence microscopy. Finally, the synergistic effect of autophagy and glutaminolysis inhibition was analyzed in an azoxymethane/dextran sodium sulfate mouse model. Results: The ATF4 pathway was activated in CRC cells upon glutaminolysis inhibition. Functionally, ATF4 transcriptionally upregulated DDIT4 to suppress mTOR, which induced pro-survival autophagy during glutaminolysis inhibition. Interestingly, glutaminolysis inhibition promoted ATF4 mRNA expression by abrogating N6-methyladenosine (m6A) modification and YTHDF2-mediated RNA decay. Finally, inhibition of ATF4-induced autophagy enhanced the antitumor efficacy of glutaminolysis inhibition. Conclusion: Glutaminolysis inhibition upregulated ATF4 expression in an m6A-dependent manner to activate pro-survival autophagy through transcriptional activation of the mTOR inhibitor DDIT4. Targeting ATF4-induced autophagy is a new strategy to synergize glutaminolysis-targeting therapies for cancer treatment.