Methylation of NRIP3 Is a Synthetic Lethal Marker for Combined PI3K and ATR/ATM Inhibitors in Colorectal Cancer.

INTRODUCTION: The aim of this study was to investigate the epigenetic regulation and underlying mechanism of NRIP3 in colorectal cancer (CRC). METHODS: Eight cell lines (SW480, SW620, DKO, LOVO, HT29, HCT116, DLD1, and RKO), 187 resected margin samples from colorectal cancer tissue, 146 cases with colorectal adenomatous polyps, and 308 colorectal cancer samples were used. Methylation-specific PCR, Western blotting, RNA interference assay, and a xenograft mouse model were used. RESULTS: NRIP3 exhibited methylation in 2.7% (5/187) of resected margin samples from colorectal cancer tissue, 32.2% (47/146) of colorectal adenomatous polyps, and 50.6% (156/308) of CRC samples, and the expression of NRIP3 was regulated by promoter region methylation. The methylation of NRIP3 was found to be significantly associated with late onset (at age 50 years or older), poor tumor differentiation, lymph node metastasis, and poor 5-year overall survival in CRC (all P < 0.05). In addition, NRIP3 methylation was an independent poor prognostic marker ( P < 0.05). NRIP3 inhibited cell proliferation, colony formation, invasion, and migration, while induced G1/S arrest. NRIP3 suppressed CRC growth by inhibiting PI3K-AKT signaling both in vitro and in vivo . Methylation of NRIP3 sensitized CRC cells to combined PI3K and ATR/ATM inhibitors. DISCUSSION: NRIP3 was frequently methylated in both colorectal adenomatous polyps and CRC. The methylation of NRIP3 may potentially serve as an early detection, late-onset, and poor prognostic marker in CRC. NRIP3 is a potential tumor suppressor. NRIP3 methylation is a potential synthetic lethal marker for combined PI3K and ATR/ATM inhibitors.

Phase 2 study of epigenetic priming with decitabine followed by cytarabine for acute myeloid leukemia in older patients.

Acute myeloid leukemia (AML) in older patients has a poor prognosis, low complete remission (CR) rates, and poor overall survival (OS). Preclinical studies have shown synergistic effects of epigenetic priming with hypomethylating agents followed by cytarabine. Based on these data, we hypothesized that an induction regimen using epigenetic priming with decitabine, followed by cytarabine would be effective and safe in older patients with previously untreated AML. Here, we conducted a phase 2 trial in which older patients with previously untreated AML received an induction regimen consisting of 1 or 2 courses of decitabine 20 mg/m2 intravenously (IV) for 5 days followed by cytarabine 100 mg/m2 continuous IV infusion for 5 days. Forty-four patients (median age 76 years) were enrolled, and CR/CRi was achieved by 26 patients (59% of all patients, 66.7% of evaluable patients). Fourteen of 21 (66.7%) patients with adverse cytogenetics achieved CR including six out of seven evaluable patients with TP53 mutations. The 4- and 8-week mortality rates were 2.3% and 9.1%, respectively, with median OS of 10.7 months. These results suggest epigenetic priming with decitabine followed by cytarabine should be considered as an option for first-line therapy in older patients with AML. This trial was registered at www.clinicaltrials.gov as # NCT01829503.

Pulmonary circulatory system characteristics are associated with future lung cancer risk.

BACKGROUND: Most of the subjects eligible for annual low-dose computed tomography (LDCT) lung screening will not develop lung cancer for their life. It is important to identify novel biomarkers that can help identify those at risk of developing lung cancer and improve the efficiency of LDCT screening programs. OBJECTIVE: This study aims to investigate the association between the morphology of the pulmonary circulatory system (PCS) and lung cancer development using LDCT scans acquired in the screening setting. METHODS: We analyzed the PLuSS cohort of 3635 lung screening patients from 2002 to 2016. Circulatory structures were segmented and quantified from LDCT scans. The time from the baseline CT scan to lung cancer diagnosis, accounting for death, was used to evaluate the prognostic ability (i.e., hazard ratio (HR)) of these structures independently and with demographic factors. Five-fold cross-validation was used to evaluate prognostic scores. RESULTS: Intrapulmonary vein volume had the strongest association with future lung cancer (HR = 0.63, p < 0.001). The joint model of intrapulmonary vein volume, age, smoking status, and clinical emphysema provided the strongest prognostic ability (HR = 2.20, AUC = 0.74). The addition of circulatory structures improved risk stratification, identifying the top 10% with 28% risk of lung cancer within 15 years. CONCLUSION: PCS characteristics, particularly intrapulmonary vein volume, are important predictors of lung cancer development. These factors significantly improve prognostication based on demographic factors and noncirculatory patient characteristics, particularly in the long term. Approximately 10% of the population can be identified with risk several times greater than average.

RASSF1A promotes ATM signaling and RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Aim: The mechanism of RASSF1A in DNA damage repair remains to be further clarified for applying to synthetic lethal strategy. Materials & methods: Eight esophageal cancer cell lines, 181 cases of esophageal dysplasia and 1066 cases of primary esophageal squamous cell carcinoma (ESCC) were employed. Methylation-specific PCR, the CRISPR/Cas9 technique, immunoprecipitation assay and a xenograft mouse model were used. Results: RASSF1A was methylated in 2.21% of esophageal dysplasia and 11.73% of ESCC. RASSF1A was also involved in DNA damage repair through activating Hippo signaling. Loss of RASSF1A expression sensitized esophageal cancer cell lines to ataxia telangiectasia mutated and rad3-related (ATR) inhibitor (VE-822) both in vitro and in vivo. Conclusion: RASSF1A methylation is a synthetic lethal marker for ATR inhibitors.

Molecular neurobiology of loss: a role for basolateral amygdala extracellular matrix.

Psychological loss is a common experience that erodes well-being and negatively impacts quality of life. The molecular underpinnings of loss are poorly understood. Here, we investigate the mechanisms of loss using an environmental enrichment removal (ER) paradigm in male rats. The basolateral amygdala (BLA) was identified as a region of interest, demonstrating differential Fos responsivity to ER and having an established role in stress processing and adaptation. A comprehensive multi-omics investigation of the BLA, spanning multiple cohorts, platforms, and analyses, revealed alterations in microglia and the extracellular matrix (ECM). Follow-up studies indicated that ER decreased microglia size, complexity, and phagocytosis, suggesting reduced immune surveillance. Loss also substantially increased ECM coverage, specifically targeting perineuronal nets surrounding parvalbumin interneurons, suggesting decreased plasticity and increased inhibition within the BLA following loss. Behavioral analyses suggest that these molecular effects are linked to impaired BLA salience evaluation, leading to a mismatch between stimulus and reaction intensity. These loss-like behaviors could be rescued by depleting BLA ECM during the removal period, helping us understand the mechanisms underlying loss and revealing novel molecular targets to ameliorate its impact.

Correlated variability in primate superior colliculus depends on functional class.

Correlated variability in neuronal activity (spike count correlations, rSC) can constrain how information is read out from populations of neurons. Traditionally, rSC is reported as a single value summarizing a brain area. However, single values, like summary statistics, stand to obscure underlying features of the constituent elements. We predict that in brain areas containing distinct neuronal subpopulations, different subpopulations will exhibit distinct levels of rSC that are not captured by the population rSC. We tested this idea in macaque superior colliculus (SC), a structure containing several functional classes (i.e., subpopulations) of neurons. We found that during saccade tasks, different functional classes exhibited differing degrees of rSC. "Delay class" neurons displayed the highest rSC, especially during saccades that relied on working memory. Such dependence of rSC on functional class and cognitive demand underscores the importance of taking functional subpopulations into account when attempting to model or infer population coding principles.

Adolescent Stress Confers Resilience to Traumatic Stress Later in Life: Role of the Prefrontal Cortex.

Background: Adolescent brains are sensitive to stressors. However, under certain circumstances, developmental stress can promote an adaptive phenotype, allowing individuals to cope better with adverse situations in adulthood, thereby contributing to resilience. Methods: Sprague Dawley rats (50 males, 48 females) were subjected to adolescent chronic variable stress (adol CVS) for 2 weeks at postnatal day 45. At postnatal day 85, a group was subjected to single prolonged stress (SPS). After a week, animals were evaluated in an auditory-cued fear conditioning paradigm, and neuronal recruitment during reinstatement was assessed by Fos expression. Patch clamp electrophysiology (17-35 cells/group) was performed in male rats to examine physiological changes associated with resilience. Results: Adol CVS blocked fear potentiation evoked by SPS. We observed that SPS impaired extinction (males) and enhanced reinstatement (both sexes) of the conditioned freezing response. Prior adol CVS prevented both effects. SPS effects were associated with a reduction of infralimbic (IL) cortex neuronal recruitment after reinstatement in males and increased engagement of the central amygdala in females, both also prevented by adol CVS, suggesting different neurocircuits involved in generating resilience between sexes. We explored the mechanism behind reduced IL recruitment in males by studying the intrinsic excitability of IL pyramidal neurons. SPS reduced excitability of IL neurons, and prior adol CVS prevented this effect. Conclusions: Our data indicate that adolescent stress can impart resilience to the effects of traumatic stress on neuroplasticity and behavior. Our data provide a mechanistic link behind developmental stress-induced behavioral resilience and prefrontal (IL) cortical excitability in males.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

Improving lung cancer diagnosis with cancer, fungal, and imaging biomarkers.

OBJECTIVE: Indeterminate pulmonary nodules (IPNs) represent a significant diagnostic burden in health care. We aimed to compare a combination clinical prediction model (Mayo Clinic model), fungal (histoplasmosis serology), imaging (computed tomography [CT] radiomics), and cancer (high-sensitivity cytokeratin fraction 21; hsCYFRA 21-1) biomarker approach to a validated prediction model in diagnosing lung cancer. METHODS: A prospective specimen collection, retrospective blinded evaluation study was performed in 3 independent cohorts with 6- to 30-mm IPNs (n = 281). Serum histoplasmosis immunoglobulin G and immunoglobulin M antibodies and hsCYFRA 21-1 levels were measured and a validated CT radiomic score was calculated. Multivariable logistic regression models were estimated with Mayo Clinic model variables, histoplasmosis antibody levels, CT radiomic score, and hsCYFRA 21-1. Diagnostic performance of the combination model was compared with that of the Mayo Clinic model. Bias-corrected clinical net reclassification index (cNRI) was used to estimate the clinical utility of a combination biomarker approach. RESULTS: A total of 281 patients were included (111 from a histoplasmosis-endemic region). The combination biomarker model including the Mayo Clinic model score, histoplasmosis antibody levels, radiomics, and hsCYFRA 21-1 level showed improved diagnostic accuracy for IPNs compared with the Mayo Clinic model alone with an area under the receiver operating characteristics curve of 0.80 (95% CI, 0.76-0.84) versus 0.72 (95% CI, 0.66-0.78). Use of this combination model correctly reclassified intermediate risk IPNs into low- or high-risk category (cNRI benign = 0.11 and cNRI malignant = 0.16). CONCLUSIONS: The addition of cancer, fungal, and imaging biomarkers improves the diagnostic accuracy for IPNs. Integrating a combination biomarker approach into the diagnostic algorithm of IPNs might decrease unnecessary invasive testing of benign nodules and reduce time to diagnosis for cancer.

Response to Neoadjuvant Targeted Therapy in Operable Head and Neck Cancer Confers Survival Benefit.

PURPOSE: Neoadjuvant targeted therapy provides a brief, preoperative window of opportunity that can be exploited to individualize cancer care based on treatment response. We investigated whether response to neoadjuvant therapy during the preoperative window confers survival benefit in patients with operable head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: A pooled analysis of treatment-naïve patients with operable HNSCC enrolled in one of three clinical trials from 2009 to 2020 (NCT00779389, NCT01218048, NCT02473731). Neoadjuvant regimens consisted of EGFR inhibitors (n = 83) or anti-ErbB3 antibody therapy (n = 9) within 28 days of surgery. Clinical to pathologic stage migration was compared with disease-free survival (DFS) and overall survival (OS) while adjusting for confounding factors using multivariable Cox regression. Circulating tumor markers validated in other solid tumor models were analyzed. RESULTS: 92 of 118 patients were analyzed; all patients underwent surgery following neoadjuvant therapy. Clinical to pathologic downstaging was more frequent in patients undergoing neoadjuvant targeted therapy compared with control cohort (P = 0.048). Patients with pathologic downstage migration had the highest OS [89.5%; 95% confidence interval (CI), 75.7-100] compared with those with no stage change (58%; 95% CI, 46.2-69.8) or upstage (40%; 95% CI, 9.6-70.4; P = 0.003). Downstage migration remained a positive prognostic factor for OS (HR, 0.22; 95% CI, 0.05-0.90) while adjusting for measured confounders. Downstage migration correlated with decreased circulating tumor markers, SOX17 and TAC1 (P = 0.0078). CONCLUSIONS: Brief neoadjuvant therapy achieved pathologic downstaging in a subset of patients and was associated with significantly better DFS and OS as well as decreased circulating methylated SOX17 and TAC1.

Preclinical Models of Chronic Stress: Adaptation or Pathology?

The experience of prolonged stress changes how individuals interact with their environment and process interoceptive cues, with the end goal of optimizing survival and well-being in the face of a now-hostile world. The chronic stress response includes numerous changes consistent with limiting further damage to the organism, including development of passive or active behavioral strategies and metabolic adjustments to alter energy mobilization. These changes are consistent with symptoms of pathology in humans, and as a result, chronic stress has been used as a translational model for diseases such as depression. While it is of heuristic value to understand symptoms of pathology, we argue that the chronic stress response represents a defense mechanism that is, at its core, adaptive in nature. Transition to pathology occurs only after the adaptive capacity of an organism is exhausted. We offer this perspective as a means of framing interpretations of chronic stress studies in animal models and how these data relate to adaptation as opposed to pathology.

Hippocampal glucocorticoid receptors modulate status epilepticus severity.

Status epilepticus (SE) is a life-threatening medical emergency with significant morbidity and mortality. SE is associated with a robust and sustained increase in serum glucocorticoids, reaching concentrations sufficient to activate the dense population of glucocorticoid receptors (GRs) expressed among hippocampal excitatory neurons. Glucocorticoid exposure can increase hippocampal neuron excitability; however, whether activation of hippocampal GRs during SE exacerbates seizure severity remains unknown. To test this, a viral strategy was used to delete GRs from a subset of hippocampal excitatory neurons in adult male and female mice, producing hippocampal GR knockdown mice. Two weeks after GR knockdown, mice were challenged with the convulsant drug pilocarpine to induce SE. GR knockdown had opposing effects on early vs late seizure behaviors, with sex influencing responses. For both male and female mice, the onset of mild behavioral seizures was accelerated by GR knockdown. In contrast, GR knockdown delayed the onset of more severe convulsive seizures and death in male mice. Concordantly, GR knockdown also blunted the SE-induced rise in serum corticosterone in male mice. GR knockdown did not alter survival times or serum corticosterone in females. To assess whether loss of GR affected susceptibility to SE-induced cell death, within-animal analyses were conducted comparing local GR knockdown rates to local cell loss. GR knockdown did not affect the degree of localized neuronal loss, suggesting cell-intrinsic GR signaling neither protects nor sensitizes neurons to acute SE-induced death. Overall, the findings reveal that hippocampal GRs exert an anti-convulsant role in both males and females in the early stages of SE, followed by a switch to a pro-convulsive role for males only. Findings reveal an unexpected complexity in the interaction between hippocampal GR activation and the progression of SE.

Epigenetic silencing of JAM3 promotes esophageal cancer development by activating Wnt signaling.

BACKGROUND: The role of JAM3 in different tumors is controversial. The epigenetic regulation and the mechanism of JAM3 remain to be elucidated in human esophageal cancer (EC). METHODS: Eleven EC cell lines, 49 cases of esophageal intraepithelial neoplasia (EIN) and 760 cases of primary EC samples were employed. Methylation-specific polymerase chain reaction, immunohistochemistry, MTT, western blot and xenograft mouse models were applied in this study. RESULTS: The inverse association between RNA expression and promoter region methylation of JAM3 was found by analyzing 185 cases of EC samples extracted from the TCGA database (p < 0.05). JAM3 was highly expressed in KYSE450, KYSE520, TE1 and YES2 cells, low level expressed in KYSE70 cells and unexpressed in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells. JAM3 was unmethylated in KYSE450, KYSE520, TE1 and YES2 cells, partial methylated in KYSE70 cells and completely methylated in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells. The expression of JAM3 is correlated with methylation status. The levels of JAM3 were unchanged in KYSE450, KYSE520, TE1 and YES2 cells, increased in KYSE70 cells and restored expression in KYSE30, KYSE150, KYSE410, KYSE510, TE13 and BIC1 cells after 5-aza-2'-deoxycytidine treatment, suggesting that the expression of JAM3 is regulated by promoter region methylation. JAM3 was methylated in 26.5% (13/49) of EIN and 51.1% (388/760) of primary EC, and methylation of JAM3 was associated significantly with tumor differentiation and family history (all p < 0.05). Methylation of JAM3 is an independent prognostic factor of poor 5-year overall survival (p < 0.05). JAM3 suppresses cell proliferation, colony formation, migration and invasion and induces G1/S arrest and apoptosis in EC. Further study demonstrated that JAM3 suppressed EC cells and xenograft tumor growth by inhibiting Wnt/ß-catenin signaling. CONCLUSION: JAM3 is frequently methylated in human EC, and the expression of JAM3 is regulated by promoter region methylation. JAM3 methylation is an early detection and prognostic marker of EC. JAM3 suppresses EC growth both in vitro and in vivo by inhibiting Wnt signaling.

Genome Sequences of Two Gordonia rubripertincta Cluster DJ Bacteriophages, Pherobrine and Burley.

Pherobrine and Burley are siphoviruses infecting Gordonia rubripertincta. Pherobrine has a 60,305-bp genome with 89 predicted protein-coding genes, and Burley has a 60,111-bp genome with 90 predicted protein-coding genes. Both phages are assigned to cluster DJ, where they share 78% gene content similarity with each other.

Differential vulnerability of anterior cingulate cortex cell types to diseases and drugs.

In psychiatric disorders, mismatches between disease states and therapeutic strategies are highly pronounced, largely because of unanswered questions regarding specific vulnerabilities of different cell types and therapeutic responses. Which cellular events (housekeeping or salient) are most affected? Which cell types succumb first to challenges, and which exhibit the strongest response to drugs? Are these events coordinated between cell types? How does disease and drug effect this coordination? To address these questions, we analyzed single-nucleus-RNAseq (sn-RNAseq) data from the human anterior cingulate cortex-a region involved in many psychiatric disorders. Density index, a metric for quantifying similarities and dissimilarities across functional profiles, was employed to identify common or salient functional themes across cell types. Cell-specific signatures were integrated with existing disease and drug-specific signatures to determine cell-type-specific vulnerabilities, druggabilities, and responsiveness. Clustering of functional profiles revealed cell types jointly participating in these events. SST and VIP interneurons were found to be most vulnerable, whereas pyramidal neurons were least. Overall, the disease state is superficial layer-centric, influences cell-specific salient themes, strongly impacts disinhibitory neurons, and influences astrocyte interaction with a subset of deep-layer pyramidal neurons. In absence of disease, drugs profiles largely recapitulate disease profiles, offering a possible explanation for drug side effects. However, in presence of disease, drug activities, are deep layer-centric and involve activating a distinct subset of deep-layer pyramidal neurons to circumvent the disease state's disinhibitory circuit malfunction. These findings demonstrate a novel application of sn-RNAseq data to explain drug and disease action at a systems level.

Lessons From a Systematic Literature Search on Diagnostic DNA Methylation Biomarkers for Colorectal Cancer: How to Increase Research Value and Decrease Research Waste?

OBJECTIVES: To improve colorectal cancer (CRC) survival and lower incidence rates, colonoscopy and/or fecal immunochemical test screening are widely implemented. Although candidate DNA methylation biomarkers have been published to improve or complement the fecal immunochemical test, clinical translation is limited. We describe technical and methodological problems encountered after a systematic literature search and provide recommendations to increase (clinical) value and decrease research waste in biomarker research. In addition, we present current evidence for diagnostic CRC DNA methylation biomarkers. METHODS: A systematic literature search identified 331 diagnostic DNA methylation marker studies published before November 2020 in PubMed, EMBASE, Cochrane Library, and Google Scholar. For 136 bodily fluid studies, extended data extraction was performed. STARD criteria and level of evidence were registered to assess reporting quality and strength for clinical translation. RESULTS: Our systematic literature search revealed multiple issues that hamper the development of DNA methylation biomarkers for CRC diagnosis, including methodological and technical heterogeneity and lack of validation or clinical translation. For example, clinical translation and independent validation were limited, with 100 of 434 markers (23%) studied in bodily fluids, 3 of 434 markers (0.7%) translated into clinical tests, and independent validation for 92 of 411 tissue markers (22%) and 59 of 100 bodily fluids markers (59%). DISCUSSION: This systematic literature search revealed that major requirements to develop clinically relevant diagnostic CRC DNA methylation markers are often lacking. To avoid the resulting research waste, clinical needs, intended biomarker use, and independent validation should be better considered before study design. In addition, improved reporting quality would facilitate meta-analysis, thereby increasing the level of evidence and enabling clinical translation.

Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells.

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Hippocampal interneurons are direct targets for circulating glucocorticoids.

The hippocampus has become a significant target of stress research in recent years because of its role in cognitive functioning, neuropathology, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the pervasive impact of stress on psychiatric and neurological disease, many of the circuit- and cell-dependent mechanisms giving rise to the limbic regulation of the stress response remain unknown. Hippocampal excitatory neurons generally express high levels of glucocorticoid receptors (GRs) and are therefore positioned to respond directly to serum glucocorticoids. These neurons are, in turn, regulated by neighboring interneurons, subtypes of which have been shown to respond to stress exposure. However, GR expression among hippocampal interneurons is not well characterized. To determine whether key interneuron populations are direct targets for glucocorticoid action, we used two transgenic mouse lines to label parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons. GR immunostaining of labeled interneurons was characterized within the dorsal and ventral dentate hilus, dentate cell body layer, and CA1 and CA3 stratum oriens and stratum pyramidale. While nearly all hippocampal SST+ interneurons expressed GR across all regions, GR labeling of PV+ interneurons showed considerable subregion variability. The percentage of PV+, GR+ cells was highest in the CA3 stratum pyramidale and lowest in the CA1 stratum oriens, with other regions showing intermediate levels of expression. Together, these findings indicate that, under baseline conditions, hippocampal SST+ interneurons are a ubiquitous glucocorticoid target, while only distinct populations of PV+ interneurons are direct targets. This anatomical diversity suggests functional differences in the regulation of stress-dependent hippocampal responses.

Technical considerations in PCR-based assay design for diagnostic DNA methylation cancer biomarkers.

BACKGROUND: DNA methylation biomarkers for early detection, risk stratification and treatment response in cancer have been of great interest over the past decades. Nevertheless, clinical implementation of these biomarkers is limited, as only < 1% of the identified biomarkers is translated into a clinical or commercial setting. Technical factors such as a suboptimal genomic location of the assay and inefficient primer or probe design have been emphasized as important pitfalls in biomarker research. Here, we use eleven diagnostic DNA methylation biomarkers for colorectal cancer (ALX4, APC, CDKN2A, MGMT, MLH1, NDRG4, SDC2, SFRP1, SFRP2, TFPI1 and VIM), previously described in a systematic literature search, to evaluate these pitfalls. RESULTS: To assess the genomic assay location, the optimal genomic locations according to TCGA data were extracted and compared to the genomic locations used in the published assays for all eleven biomarkers. In addition, all primers and probes were technically evaluated according to several criteria, based on literature and expert opinion. Both assay location and assay design quality varied widely among studies. CONCLUSIONS: Large variation in both assay location and design hinders the development of future DNA methylation biomarkers as well as inter-study comparability.

Microsaccades as a marker not a cause for attention-related modulation.

Recent evidence suggests that microsaccades are causally linked to the attention-related modulation of neurons-specifically, that microsaccades toward the attended location are required for the subsequent changes in firing rate. These findings have raised questions about whether attention-related modulation is due to different states of attention as traditionally assumed or might instead be a secondary effect of microsaccades. Here, in two rhesus macaques, we tested the relationship between microsaccades and attention-related modulation in the superior colliculus (SC), a brain structure crucial for allocating attention. We found that attention-related modulation emerged even in the absence of microsaccades, was already present prior to microsaccades toward the cued stimulus, and persisted through the suppression of activity that accompanied all microsaccades. Nonetheless, consistent with previous findings, we also found significant attention-related modulation when microsaccades were directed toward, rather than away from, the cued location. Thus, despite the clear links between microsaccades and attention, microsaccades are not necessary for attention-related modulation, at least not in the SC. They do, however, provide an additional marker for the state of attention, especially at times when attention is shifting from one location to another.