Intermediate Phenotypes Research Articles

Characterization of HIV variants from paired Cerebrospinal fluid and Plasma samples in primary microglia and CD4+ T-cells.

Despite antiretroviral therapy (ART), HIV persistence in the central nervous system (CNS) continues to cause a range of cognitive impairments in people living with HIV (PLWH). Upon disease progression, transmigrating CCR5-using T-cell tropic viruses are hypothesized to evolve into macrophage-tropic viruses in the CNS that can efficiently infect low CD4-expressing cells, such as microglia. We examined HIV-1 RNA concentration, co-receptor usage, and CSF compartmentalization in paired CSF and blood samples from 19 adults not on treatment. Full-length envelope CSF- and plasma-derived reporter viruses were generated from 3 subjects and phenotypically characterized in human primary CD4+ T-cells and primary microglia. Median HIV RNA levels were higher in plasma than in CSF (5.01 vs. 4.12 log10 cp/mL; p = 0.004), and coreceptor usage was mostly concordant for CCR5 across the paired samples (n = 17). Genetically compartmentalized CSF viral populations were detected in 2 subjects, one with and one without neurological symptoms. All viral clones could replicate in T-cells (R5 T cell-tropic). In addition, 3 CSF and 1 plasma patient-derived viral clones also had the capacity to replicate in microglia/macrophages and, therefore have an intermediate macrophage tropic phenotype. Overall, with this study, we demonstrate that in a subset of PLWH, plasma-derived viruses undergo genetic and phenotypic evolution within the CNS, indicating viral infection and replication in CNS cells. It remains to be studied whether the intermediate macrophage-tropic phenotype observed in primary microglia represents a midpoint in the evolution towards a macrophage-tropic phenotype that can efficiently replicate in microglial cells and propagate viral infection in the CNS.

Unveiling Promising Neuroimaging Biomarkers for Schizophrenia Through Clinical and Genetic Perspectives.

Schizophrenia is a complex and serious brain disorder. Neuroscientists have become increasingly interested in using magnetic resonance-based brain imaging-derived phenotypes (IDPs) to investigate the etiology of psychiatric disorders. IDPs capture valuable clinical advantages and hold biological significance in identifying brain abnormalities. In this review, we aim to discuss current and prospective approaches to identify potential biomarkers for schizophrenia using clinical multimodal neuroimaging and imaging genetics. We first described IDPs through their phenotypic classification and neuroimaging genomics. Secondly, we discussed the applications of multimodal neuroimaging by clinical evidence in observational studies and randomized controlled trials. Thirdly, considering the genetic evidence of IDPs, we discussed how can utilize neuroimaging data as an intermediate phenotype to make association inferences by polygenic risk scores and Mendelian randomization. Finally, we discussed machine learning as an optimum approach for validating biomarkers. Together, future research efforts focused on neuroimaging biomarkers aim to enhance our understanding of schizophrenia.

Genetic variation in UGT1A1 is not associated with altered liver biochemical parameters in healthy volunteers participating in bioequivalence trials

Introduction: Bioequivalence clinical trials are conducted in healthy volunteers whose blood tests should be within normal limits; individuals with Gilbert syndrome (GS) are excluded from these studies on suspicion of any liver disease, even if the change is clinically insignificant. GS is a benign genetic disorder characterized by elevated bilirubin levels, the primary cause of which is the presence of polymorphisms in UGT1A1 gene. In this work, subjects with UGT1A1 intermediate (IM) or poor (PM) metabolizer genotype-informed phenotypes were investigated to determine whether they have a higher incidence of liver disease or other biochemical parameters.Methods: The study population comprised 773 healthy volunteers who underwent biochemical analysis at baseline and at the end of the study which were genotyped for UGT1A1*80 (rs887829), as an indicator of UGT1A1*80+*28 (rs887829 and rs3064744), and UGT1A1*6 (rs4148323).Results: Bilirubin levels were higher in subjects IMs and PMs compared to normal metabolizers (NMs). Decreased uric acid levels was observed in PMs compared to NMs. No associations were observed in liver enzyme levels according to UGT1A1 phenotype.Discussion: Considering that there is no hepatic toxicity in subjects with UGT1A1 IM or PM phenotype, who are more likely to develop GS, this study suggests that they could be included in bioequivalence clinical trials as their biochemical parameters are not affected outside normal ranges.

Abstract PO5-09-02: Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density

Abstract Introduction: Breast cancer incidence is rising in premenopausal, hence, there is a critical need to understand factors underlying premenopausal breast cancer development in order to guide targeted prevention. Mammographic breast density is a strong risk factor for, as well as an intermediate phenotype for premenopausal breast cancer. Yet, the molecular mechanisms underlying the associations of dense breasts with breast cancer are not well understood. Our objectives in this study are to perform proteomic analysis in breast tissues to (i) identify proteins that are associated with breast cancer development in premenopausal women; (ii) determine which of these proteins are also associated with dense breasts. Methods: We performed proteomic analysis on tumor and adjacent normal tissues from 50 premenopausal women with breast cancer who had breast tissue samples archived at the St. Louis Breast Tumor Registry. Samples were analyzed on Orbitrap Eclipse Tribrid coupled with Vanquish Neo LC system (Thermo Fisher Scientific). Database search was performed using MaxQuant and MS1 LFQ intensities were used for further data analysis. We performed multiple imputation using Bayesian Principal Component Analysis and hierarchical clustering analysis using ‘ConsensusClusterPlus’ package in R. Pathway enrichment analysis was performed using Reactome Pathway database. 10 times repeated 5-fold cross validation using Logistic Regression (LR) was performed for model development. Top 10 proteins were selected by variable importance in logistic regression, and recursive feature elimination was performed for feature selection. We corrected for multiple testing by setting the false discovery rate, FDR, p-value < 0.05. Results: After imputing for missing values, we identified 3,571 proteins that were expressed in tumor and normal breast tissues. We selected 1,640 proteins for further analysis using median absolute deviation analysis. In multivariable-adjusted analysis, 451 proteins were up-regulated, and 180 proteins were down-regulated in tumor tissues compared to adjacent normal tissues (FDR < 0.05). 53 of the 451 proteins that were upregulated in tumor tissues were also upregulated in dense breasts. Some of the top proteins that were upregulated in both tumor tissues and dense breasts were AIMP2, ALDH18A1, BZW1, CKAP5, COPG1, ERGIC1, GSPT1, ILF2, IPO7, LRBA, and PSMD12. Mammographic breast density protein clusters were highly correlated with hormone receptor positivity clusters and were enriched in ESR-mediated signaling, IGF1R signaling, NOTCH4, PTEN regulation, and NFkB pathway. Discussion: Using untargeted proteomics, we identified proteomic signatures of breast cancer development in premenopausal women. We further identified protein biomarkers and pathways that are shared between breast tumors and dense breast tissues in premenopausal women. Our novel findings highlight mammographic breast density as a strong intermediate phenotype that can be targeted in breast cancer prevention in premenopausal women. Larger studies are needed to validate our findings. Citation Format: Shaili Tapiavala, Minsoo Son, Graham Colditz, Ah Young Goo, Adetunji Toriola. Breast Tissue Proteomic Profile of Breast Cancer in Premenopausal Women and Association with Mammographic Breast Density [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-02.

Attention-deficit/hyperactivity disorder combined subtype exacerbates opioid use disorder consequences: Mediation by impulsive phenotypes

BackgroundAttention-deficit/hyperactivity disorder (ADHD) is highly prevalent and associated with opioid use disorder (OUD). Yet, little is known about the mechanisms by which ADHD (which is a heterogeneous construct/diagnosis) might alter the trajectory of OUD outcomes in persons who use heroin. AimWe examined whether ADHD subtypes are related to heroin-use consequences and the extent to which the effects of ADHD on lifetime heroin-use consequences are mediated by two impulsivity factors that may be partly independent of ADHD: foreshortened time perspective and drug-use impulsivity. MethodsIndividuals who reported regular heroin use (N=250) were screened using the Assessment of Hyperactivity and Attention (AHA), Impulsive Relapse Questionnaire (IRQ), Stanford Time Perception Inventory (STPI), and a comprehensive assessment of lifetime and current substance use and substance-related consequences. This secondary analysis examined whether ADHD or intermediate phenotypes predicted heroin-use consequences. ResultsRelative to participants whose AHA scores indicated lifetime absence of ADHD (n=88), those with scores indicating persistent ADHD (childhood and adult, n=62) endorsed significantly more total lifetime heroin-use consequences despite comparable heroin-use severity. Likewise, there was a significant indirect effect of the combined ADHD subtype in childhood on lifetime heroin-use consequences. This effect was mediated by STPI scores indicating less future (and more hedonism in the present) temporal orientation and by IRQ scores indicating less capacity for delaying drug use. ConclusionThe combined ADHD subtype is significantly associated with lifetime heroin-use consequences, and this effect is mediated through higher drug-use impulsivity (less capacity for delay) and lower future temporal orientation.

Abstract LB324: STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas

Abstract Unresolved chronic inflammation has been postulated to drive prostate cancer development. We have proposed that proliferative inflammatory atrophy (PIA) can lead to the development of preneoplastic high grade prostatic intraepithelial neoplasia (PIN) lesions and/or directly to adenocarcinoma at times. Additionally, some PIA cells harbor molecular alterations seen in PIN and invasive carcinoma lesions, with a low frequency of PIA cells showing MYC over-expression, partial promoter hypermethylation and silencing of GSTP1, and formation of ETS gene fusions. PIA appears to result from inflammatory injury and regeneration in which proliferative and atrophic epithelial cells have an intermediate cell phenotype with both luminal and basal cell features, including basal cell markers, keratin 5 and BCL2, as well as varying levels of prostate luminal enriched genes such as androgen receptor, PSA and NKX3.1. Also, a number of gene products that are apparently stress induced, which are not expressed in normal prostate are induced in at least a subset of intermediate luminal cells in PIA, including GSTA1, PTGS2, LTF and PIGR (PMIDs:17384581, 37550827). Stimulator of interferon genes (STING), encoded by TMEM173, is a multipass transmembrane ER associated protein, and is a critical hub for innate responses directed against numerous bacterial, viral and parasitic pathogens, and is important for the induction and maintenance of adaptive immunity in a number of situations. Here, we examined STING expression by IHC, using a genetically validated assay, and show that the majority of atrophic prostatic luminal epithelial cells in all PIA lesions (N=20) consistently express high levels of STING, which is present in normal prostatic basal epithelial cells yet absent in normal-appearing prostatic luminal cells. In primary prostatic adenocarcinomas (> 260 tumors), STING protein was either completely negative , or extremely low in ~ 95% of cases. That STING in PIA is activate is supported by single cell RNA sequencing (scRNAseq) data from human prostatectomy tissues that show cells with the phenotype of intermediate luminal cells, indicative of PIA, express a number of interferon response genes that are not expressed in normal luminal cells (PMID 37905029). To our knowledge this is the first systematic report of STING protein staining in human prostate cancer and benign tissues. We postulate that upregulation of STING in human intermediate cells in PIA represents an inflammatory response that can drive interferon stimulated gene expression and inflammation in PIA. This innate immune response is absent, potentially by gene silencing, in prostatic adenocarcinoma, which may help neoplastic cells evade the immune response and could help contribute to the long standing finding that most prostate cancers are immunologically cold. Citation Format: Thomas M. Steele, Alan K. Meeker, Mindy K. Graham, Rulin Wang, Carolina Gomes-Alexandre, William G. Nelson, Fernanda Caramella Pereiria, Sushant Kachhap, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. STING protein expression is increased in intermediate luminal cells in proliferative inflammatory atrophy of the prostate (PIA) and is absent in the majority of primary prostatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB324.

Where east meets west: Phylogeography of the high Arctic North American brant goose.

Genetic variation in Arctic species is often influenced by vicariance during the Pleistocene, as ice sheets fragmented the landscape and displaced populations to low- and high-latitude refugia. The formation of secondary contact or suture zones during periods of ice sheet retraction has important consequences on genetic diversity by facilitating genetic connectivity between formerly isolated populations. Brant geese (Branta bernicla) are a maritime migratory waterfowl (Anseriformes) species that almost exclusively uses coastal habitats. Within North America, brant geese are characterized by two phenotypically distinct subspecies that utilize disjunct breeding and wintering areas in the northern Pacific and Atlantic. In the Western High Arctic of Canada, brant geese consist of individuals with an intermediate phenotype that are rarely observed nesting outside this region. We examined the genetic structure of brant geese populations from each subspecies and areas consisting of intermediate phenotypes using mitochondrial DNA (mtDNA) control region sequence data and microsatellite loci. We found a strong east-west partition in both marker types consistent with refugial populations. Within subspecies, structure was also observed at mtDNA while microsatellite data suggested the presence of only two distinct genetic clusters. The Western High Arctic (WHA) appears to be a secondary contact zone for both Atlantic and Pacific lineages as mtDNA and nuclear genotypes were assigned to both subspecies, and admixed individuals were observed in this region. The mtDNA sequence data outside WHA suggests no or very restricted intermixing between Atlantic and Pacific wintering populations which is consistent with published banding and telemetry data. Our study indicates that, although brant geese in the WHA are not a genetically distinct lineage, this region may act as a reservoir of genetic diversity and may be an area of high conservation value given the potential of low reproductive output in this species.

The overlap across psychotic disorders: A functional network connectivity analysis

Functional network connectivity (FNC) has previously been shown to distinguish patient groups from healthy controls (HC). However, the overlap across psychiatric disorders such as schizophrenia (SZ), bipolar (BP), and schizoaffective disorder (SAD) is not evident yet. This study focuses on studying the overlap across these three psychotic disorders in both dynamic and static FNC (dFNC/sFNC). We used resting-state fMRI, demographics, and clinical information from the Bipolar–Schizophrenia Network on Intermediate Phenotypes cohort (BSNIP). The data includes three groups of patients with schizophrenia (SZ, N = 181), bipolar (BP, N = 163), and schizoaffective (SAD, N = 130) and HC (N = 238) groups. After estimating each individual's dFNC, we group them into three distinct states. We evaluated two dFNC features, including occupancy rate (OCR) and distance travelled over time. Finally, the extracted features, including both sFNC and dFNC, are tested statistically across patients and HC groups. In addition, we explored the link between the clinical scores and the extracted features. We evaluated the connectivity patterns and their overlap among SZ, BP, and SAD disorders (false discovery rate or FDR corrected p < 0.05). Results showed dFNC captured unique information about overlap across disorders where all disorder groups showed similar pattern of activity in state 2. Moreover, the results showed similar patterns between SZ and SAD in state 1 which was different than BP. Finally, the distance travelled feature of SZ (average R = 0.245, p < 0.01) and combined distance travelled from all disorders was predictive of the PANSS symptoms scores (average R = 0.147, p < 0.01).

Co-Expression Network Analysis Unveiled lncRNA-mRNA Links Correlated to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor Resistance and/or Intermediate Epithelial-to-Mesenchymal Transition Phenotypes in a Human Non-Small Cell Lung Cancer Cellular Model System.

We investigated mRNA-lncRNA co-expression patterns in a cellular model system of non-small cell lung cancer (NSCLC) sensitive and resistant to the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib/gefitinib. The aim of this study was to unveil insights into the complex mechanisms of NSCLC targeted therapy resistance and epithelial-to-mesenchymal transition (EMT). Genome-wide RNA expression was quantified for weighted gene co-expression network analysis (WGCNA) to correlate the expression levels of mRNAs and lncRNAs. Functional enrichment analysis and identification of lncRNAs were conducted on modules associated with the EGFR-TKI response and/or intermediate EMT phenotypes. We constructed lncRNA-mRNA co-expression networks and identified key modules and their enriched biological functions. Processes enriched in the selected modules included RHO (A, B, C) GTPase and regulatory signaling pathways, apoptosis, inflammatory and interleukin signaling pathways, cell adhesion, cell migration, cell and extracellular matrix organization, metabolism, and lipid metabolism. Interestingly, several lncRNAs, already shown to be dysregulated in cancer, are connected to a small number of mRNAs, and several lncRNAs are interlinked with each other in the co-expression network.

Preference for Immediate Rewards in Attention-Deficit/Hyperactivity Disorder and Substance Use Disorder: A Shared Intermediate Phenotype?

Abstract Purpose of Review This brief review explores to what extent ADHD and SUD are both associated with reward-related impulsivity, operationalized as steep delay discounting (DD). However, an integrated view on DD as a potentially shared intermediate phenotype for these frequently co-occurring conditions is lacking. Recent Findings Though mostly studied in separate conditions, some studies have investigated DD specifically in patients with co-occurring conditions of SUD and ADHD. In addition to reviewing literature that has documented the associations between ADHD and relatively steep DD and between SUD and relatively steep DD, we also reviewed more recent literature in which steep DD has been examined as a transdiagnostic mechanism. Summary Overall, literature on DD in SUD suggests a dose-response relation between SUD severity and DD. The literature suggests that relatively steep DD is a mechanism associated to (ab)use of a range of substances. As for the link between DD and ADHD, several meta-analyses reported steeper DD in individuals with ADHD than controls (medium effect sizes). Because these bodies of literature are based on studies in which the influence of comorbidity was not assessed, it is hard to conclude that relatively steep DD is directly related to the ADHD symptoms per se, or to the SUD per se. Therefore, a close inspection of more recent literature in which comorbidity was taken into account, and DD was treated as a transdiagnostic mechanism, was included here. Based on this reviewed recent work, we propose a model about the nature of the co-occurrence of ADHD, SUD, and DD. Implications of the nature of co-occurring ADHD, SUD, and relatively steep DD for prevention and intervention strategies are discussed.

Identification of Siglec-1-negative alveolar macrophages with pro-inflammatory phenotypes in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD) orchestrate persistent inflammation in the airway. However, sub-populations of AMs participating in the chronic inflammation have been poorly characterized. We previously reported that Siglec-1 expression on AMs, which is important for bacteria engulfment, was decreased in COPD. Here, we show that Siglec-1-negative AMs isolated from COPD lung tissues exhibit a pro-inflammatory phenotype and is associated with poor clinical outcomes in patients with COPD. Using flow-cytometry, we segregated three subsets of AMs based on the expression of Siglec-1 and their side scattergram (SSC) and forward scattergram (FSC) properties: Siglec-1+SSChiFSChi, Siglec-1-SSChiFSChi and Siglec-1-SSCloFSClo subsets. The Siglec-1-SSCloFSClo subset number was increased in COPD. RNA-sequencing revealed upregulation of multiple pro-inflammatory signaling pathways and emphysema-associated matrix metalloproteases in the Siglec-1-SSCloFSClo subset. Gene set enrichment analysis indicated that the Siglec-1-SSCloFSClo subset adopted intermediate phenotypes between monocytes and mature alveolar macrophages. Functionally, these cells produced TNF-α, IL-6 and IL-8 at baseline, and these cytokines were significantly increased in response to viral RNA. The increase in Siglec-1-negative AMs in induced sputum is associated with future exacerbation risk and lung function decline in patients with COPD. Collectively, the novel Siglec-1-SSCloFSClo subset of AMs display pro-inflammatory properties, and their emergence in COPD airways may be associated with poor clinical outcomes.

A Variety of Mouse PYHIN Proteins Restrict Murine and Human Retroviruses.

PYHIN proteins are only found in mammals and play key roles in the defense against bacterial and viral pathogens. The corresponding gene locus shows variable deletion and expansion ranging from 0 genes in bats, over 1 in cows, and 4 in humans to a maximum of 13 in mice. While initially thought to act as cytosolic immune sensors that recognize foreign DNA, increasing evidence suggests that PYHIN proteins also inhibit viral pathogens by more direct mechanisms. Here, we examined the ability of all 13 murine PYHIN proteins to inhibit HIV-1 and murine leukemia virus (MLV). We show that overexpression of p203, p204, p205, p208, p209, p210, p211, and p212 strongly inhibits production of infectious HIV-1; p202, p207, and p213 had no significant effects, while p206 and p214 showed intermediate phenotypes. The inhibitory effects on infectious HIV-1 production correlated significantly with the suppression of reporter gene expression by a proviral Moloney MLV-eGFP construct and HIV-1 and Friend MLV LTR luciferase reporter constructs. Altogether, our data show that the antiretroviral activity of PYHIN proteins is conserved between men and mice and further support the key role of nuclear PYHIN proteins in innate antiviral immunity.

Abstract 1933: Dissecting acquired resistance to KRASG12D inhibition in a mouse model of pancreatic ductal adenocarcinoma

Abstract KRAS is a major oncogenic driver in pancreatic ductal adenocarcinoma (PDAC) and is mutationally activated in approximately 90% of cases, with the glycine-to-aspartic acid substitution at position 12 (p.G12D) being the most prevalent alteration. Mutations in this oncogene have been associated with more aggressive disease and poorer outcomes and have remained “undruggable” for more than three decades. However, the recent development of mutant-specific small molecule inhibitors of KRAS has given hope for the treatment of this highly lethal and treatment-refractory malignancy. Here we evaluated the genomic and transcriptional mechanisms of resistance to MRTX1133, a first-in-class inhibitor of KRASG12D, in an autochthonous mouse model of PDAC. We leveraged single-nucleus RNA sequencing and whole-exome sequencing on 17 tumors from KPC mouse specimens (LSL-KrasG12D/+; LSL-Trp53R172H/+; p48-Cre) that were treated with either MRTX1133 or the vehicle, including 6 samples from mice that developed acquired resistance to KRASG12D inhibition. Our analysis has uncovered that resistance is multifaceted and characterized by both emergent genomic and transcriptional features. Genetically, resistant tumors harbored clonal and subclonal amplifications in the HIPPO pathway, cell cycle regulators, and ABC transporters. Transcriptionally, we employed non-negative matrix factorization and identified recurrent gene expression programs in resistant and vehicle samples. Using this approach, we uncovered the existence of an intermediate phenotype between classical and mesenchymal cell states that is enriched in tumors with evolved resistance to MRTX1133. In summary, our study provides a high-resolution genomic and transcriptional landscape of resistance to KRAS targeting in a preclinical in vivo model of PDAC, highlighting the complexity of treatment resistance and identifying various pathways and effectors that may serve as potential new targets for combination therapy. Citation Format: Julien Dilly, Laleh Abbassi, Connor J. Hennessey, Giselle A. Uribe, Brendan Parent, Annan Yang, Kevin S. Kapner, Ziyue Li, Kyle E. Evans, Shatavisha Dasgupta, Megan T. Hoffman, Li Qiang, Felix P. Hambitzer, Seema Chugh, Alex K. Shalek, Stephanie K. Dougan, Brian M. Wolpin, Jonathan A. Nowak, Srivatsan Raghavan, Peter S. Winter, Andrew J. Aguirre. Dissecting acquired resistance to KRASG12D inhibition in a mouse model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1933.

CDC2 plasticity and acquisition of a DC3-like phenotype mediated by IL-6 and PGE2 in a patient-derived colorectal cancer organoids model.

Metastatic colorectal cancer (CRC) is highly resistant to therapy and prone to recur. The tumor-induced local and systemic immunosuppression allows cancer cells to evade immunosurveillance, facilitating their proliferation and dissemination. Dendritic cells (DCs) are required for the detection, processing, and presentation of tumor antigens, and subsequently for the activation of antigen-specific T cells to orchestrate an effective antitumor response. Notably, successful tumors have evolved mechanisms to disrupt and impair DC functions, underlining the key role of tumor-induced DC dysfunction in promoting tumor growth, metastasis initiation, and treatment resistance. Conventional DC type 2 (cDC2) are highly prevalent in tumors and have been shown to present high phenotypic and functional plasticity in response to tumor-released environmental cues. This plasticity reverberates on both the development of antitumor responses and on the efficacy of immunotherapies in cancer patients. Uncovering the processes, mechanisms, and mediators by which CRC shapes and disrupts cDC2 functions is crucial to restoring their full antitumor potential. In this study, we use our recently developed 3D DC-tumor co-culture system to investigate how patient-derived primary and metastatic CRC organoids modulate cDC2 phenotype and function. We first demonstrate that our collagen-based system displays extensive interaction between cDC2 and tumor organoids. Interestingly, we show that tumor-corrupted cDC2 shift toward a CD14+ population with defective expression of maturation markers, an intermediate phenotype positioned between cDC2 and monocytes, and impaired T-cell activating abilities. This phenotype aligns with the newly defined DC3 (CD14+ CD1c+ CD163+) subset. Remarkably, a comparable population was found to be present in tumor lesions and enriched in the peripheral blood of metastatic CRC patients. Moreover, using EP2 and EP4 receptor antagonists and an anti-IL-6 neutralizing antibody, we determined that the observed phenotype shift is partially mediated by PGE2 and IL-6. Importantly, our system holds promise as a platform for testing therapies aimed at preventing or mitigating tumor-induced DC dysfunction. Overall, our study offers novel and relevant insights into cDC2 (dys)function in CRC that hold relevance for the design of therapeutic approaches.

Effect of L-Methylfolate Supplementation on Sleep for Patients with Reduced Methylenetetrahydrofolate Reductase Activity

Introduction Clinicians have limited options outside controlled substances to address sleep disturbance, which left untreated can negatively affect patient outcomes in cardiovascular health, mental health, immunologic function, and more. For some, genetic factors may influence sleep disturbances. L-methylfolate, the active form of folate, plays a critical role in regulation of monoamine neurotransmitters known to have significant impact on sleep regulation: dopamine, serotonin, norepinephrine. Single nucleotide polymorphisms of the enzyme methylene-tetrahydrofolate-reductase are common and can impact monoamine production. The goal of this study was to evaluate effects of L-methylfolate supplementation on sleep in a cohort with reduced methylene tetrahydrofolate reductase (MTHFR) activity. Methods A retrospective cohort of patients being treated with L-methylfolate in a concierge medical clinic setting was studied. Patients presenting with sleep complaints were evaluated using the Patient-Reported Outcomes Measurement Information System at baseline. Patients with known MTHFR polymorphisms at either C667T and/or A1298C were recommended 5 mg of L-methylfolate daily and were reevaluated at 2 wks, at 4 wks, and at 8 wks of supplementation. Statistical comparisons were made utilizing ANOVA and T-test comparisons. Results Ten were included in the final cohort: six male and four female, average age 43 ± 16 years. Beginning at wk 2, average sleep disturbance improved significantly by −6.94 points (p = 0.005) and by 8 wks, all patients had improvement with a −14.34 change in disturbance from baseline (p = 0.001). Conclusion Improvement in sleep disturbance was seen in both low and intermediate function phenotypes. L-methylfolate may be useful for improving sleep in patients with MTHFR polymorphism.

Beyond Quiescent and Active: Intermediate Microglial Transcriptomic States in a Mouse Model of Down Syndrome.

Research on microglia in Down syndrome (DS) has shown that microglial activation, increased inflammatory gene expression, and oxidative stress occur at different ages in DS brains. However, most studies resulted in simplistic definitions of microglia as quiescent or active, ignoring potential intermediate states. Indeed, recent work on microglial cells in young DS brains indicated that those evolve through different intermediate activation phenotypes before reaching a fully activated state. Here we used single nucleus RNA sequencing, to examine how trisomy affects microglial states in the Ts65Dn mouse model of DS. Despite no substantial changes in the proportion of glial populations, differential expression analysis revealed cell type-specific gene expression changes, most notably in astroglia, microglia, and oligodendroglia. Focusing on microglia, we identified differential expression of genes associated with different microglial states, including disease-associated microglia (DAMs), activated response microglia (ARMs), and human Alzheimer's disease microglia (HAMs), in trisomic microglia. Furthermore, pseudotime analysis reveals a unique reactivity profile in Ts65Dn microglia, with fewer in a homeostatic state and more in an intermediate aberrantly reactive state than in euploid microglia. This comprehensive understanding of microglial transcriptional dynamics sheds light on potential pathogenetic mechanisms but also possible avenues for therapy for neurodevelopmental disorders.

Pneumococcal Meningitis Induces Hearing Loss and Cochlear Ossification Modulated by Chemokine Receptors CX3CR1 and CCR2.

Pneumococcal meningitis is a major cause of hearing loss and permanent neurological impairment despite widely available antimicrobial therapies to control infection. Methods to improve hearing outcomes for those who survive bacterial meningitis remains elusive. We used a mouse model of pneumococcal meningitis to evaluate the impact of mononuclear phagocytes on hearing outcomes and cochlear ossification by altering the expression of CX3CR1 and CCR2 in these infected mice. We induced pneumococcal meningitis in approximately 500 C57Bl6 adult mice using live Streptococcus pneumoniae (serotype 3, 1 × 105 colony forming units (cfu) in 10µl) injected directly into the cisterna magna of anesthetized mice and treated these mice with ceftriaxone daily until recovered. We evaluated hearing thresholds over time, characterized the cochlear inflammatory response, and quantified the amount of new bone formation during meningitis recovery. We used microcomputed tomography (microCT) scans to quantify cochlear volume loss caused by neo-ossification. We also performed perilymph sampling in live mice to assess the integrity of the blood-perilymph barrier during various time intervals after meningitis. We then evaluated the effect of CX3CR1 or CCR2 deletion in meningitis symptoms, hearing loss, macrophage/monocyte recruitment, neo-ossification, and blood labyrinth barrier function. Sixty percent of mice with pneumococcal meningitis developed hearing loss. Cochlear fibrosis could be detected within 4days of infection, and neo-ossification by 14days. Loss of spiral ganglion neurons was common, and inner ear anatomy was distorted by scarring caused by new soft tissue and bone deposited within the scalae. The blood-perilymph barrier was disrupted at 3days post infection (DPI) and was restored by seven DPI. Both CCR2 and CX3CR1 monocytes and macrophages were present in the cochlea in large numbers after infection. Neither chemokine receptor was necessary for the induction of hearing loss, cochlear fibrosis, ossification, or disruption of the blood-perilymph barrier. CCR2 knockout (KO) mice suffered the most severe hearing loss. CX3CR1 KO mice demonstrated an intermediate phenotype with greater susceptibility to hearing loss compared to control mice. Elimination of CX3CR1 mononuclear phagocytes during the first 2weeks after meningitis in CX3CR1-DTR transgenic mice did not protect mice from any of the systemic or hearing sequelae of pneumococcal meningitis. Pneumococcal meningitis can have devastating effects on cochlear structure and function, although not all mice experienced hearing loss or cochlear damage. Meningitis can result in rapid progression of hearing loss with fibrosis starting at four DPI and ossification within 2weeks of infection detectable by light microscopy. The inflammatory response to bacterial meningitis is robust and can affect all three scalae. Our results suggest that CCR2 may assist in controlling infection and maintaining cochlear patency, as CCR2 knockout mice experienced more severe disease, more rapid hearing loss, and more advanced cochlear ossification after pneumococcal meningitis. CX3CR1 also may play an important role in the maintenance of cochlear patency.

A dynamic model of inorganic arsenic-induced carcinogenesis reveals an epigenetic mechanism for epithelial–mesenchymal plasticity.

Inorganic arsenic (iAs) causes cancer by initiating dynamic transitions between epithelial and mesenchymal cell phenotypes. These transitions transform normal cells into cancerous cells, and cancerous cells into metastatic cells. Most in vitro models assume that transitions between states are binary and complete, and do not consider the possibility that intermediate, stable cellular states might exist. In this paper, we describe a new, two-hit in vitro model of iAs-induced carcinogenesis that extends to 28 weeks of iAs exposure. Through week 17, the model faithfully recapitulates known and expected phenotypic, genetic, and epigenetic characteristics of iAs-induced carcinogenesis. By 28 weeks, however, exposed cells exhibit stable, intermediate phenotypes and epigenetic properties, and key transcription factor promoters (SNAI1, ZEB1) enter an epigenetically poised or bivalent state. These data suggest that key epigenetic transitions and cellular states exist during iAs-induced epithelial-to-mesenchymal transition (EMT), and that it is important for our in vitro models to encapsulate all aspects of EMT and the mesenchymal-to-epithelial transition (MET). In so doing, and by understanding the epigenetic systems controlling these transitions, we might find new, unexpected opportunities for developing targeted, cell state-specific therapeutics.

Feeding selectivity and a functional trade-off in a benthic fish with a continuous morphological variation: an experimental test

BackgroundFunctional trade-offs through ecological specializations are hypothesized to become causes of adaptive phenotypic divergence under divergent natural selection, where intermediate phenotypes may have the lowest fitness. Evidence of phenotypic divergence in a trade-off between populations experiencing different environmental/ecological conditions is abundant. However, traits in divergent selection sometimes present non-discrete (unimodal) variability, including intermediate phenotypes, although the underlying mechanisms are poorly documented. A benthic cyprinid fish, Pseudogobio esocinus, in Lake Biwa, central Japan, exhibits a large non-discrete/continuous variation in mouthpart morphology (from wide to narrow) within a lake population. The variation is linked with individual diets (i.e., the compositions of two different types of prey) even at a single site, and thus the variability is hypothesized to persist under divergent selection for prey usage. As a first step toward understanding the persistence mechanisms, here I examined the presence of morphology-dependent feeding selectivity and a functional trade-off in a laboratory experiment.ResultsWhen each experimental fish was simultaneously provided the different types of prey (chironomid larvae and amphipods), the fish mostly utilized chironomid larvae as primary prey. However, compared with the wider-mouthed fish, the narrower-mouthed fish took a larger proportion of amphipods as secondary prey by changing feeding (attacking) behavior. The intermediate-mouthed fish had lower feeding efficiency than the extreme-mouthed fish, indicating potential disadvantage of the intermediate phenotype.ConclusionsThis experimental result supports the presence of morphology-dependent feeding performance and a functional trade-off with potential impacts on trait variability, which may favor specializations rather than generalizations. In the wild, however, there may be some situations for relaxing the trade-off, such as temporally fluctuating prey availability that could also favor generalizations depending on the conditions, and thus, both extreme and intermediate phenotypes may persist/coexist in a single habitat. Although further examinations, especially focusing on feeding efficiency for each prey type separated from the effects of prey selectivity, are needed, this case represents an opportunity to consider the possible mechanisms of the persistence of phenotypic variation that is maintained without divergence even in a trade-off.

Enlarged pituitary gland volume: a possible state rather than trait marker of psychotic disorders.

Enlarged pituitary gland volume could be a marker of psychotic disorders. However, previous studies report conflicting results. To better understand the role of the pituitary gland in psychosis, we examined a large transdiagnostic sample of individuals with psychotic disorders. The study included 751 participants (174 with schizophrenia, 114 with schizoaffective disorder, 167 with psychotic bipolar disorder, and 296 healthy controls) across six sites in the Bipolar-Schizophrenia Network on Intermediate Phenotypes consortium. Structural magnetic resonance images were obtained, and pituitary gland volumes were measured using the MAGeT brain algorithm. Linear mixed models examined between-group differences with controls and among patient subgroups based on diagnosis, as well as how pituitary volumes were associated with symptom severity, cognitive function, antipsychotic dose, and illness duration. Mean pituitary gland volume did not significantly differ between patients and controls. No significant effect of diagnosis was observed. Larger pituitary gland volume was associated with greater symptom severity (F = 13.61, p = 0.0002), lower cognitive function (F = 4.76, p = 0.03), and higher antipsychotic dose (F = 5.20, p = 0.02). Illness duration was not significantly associated with pituitary gland volume. When all variables were considered, only symptom severity significantly predicted pituitary gland volume (F = 7.54, p = 0.006). Although pituitary volumes were not increased in psychotic disorders, larger size may be a marker associated with more severe symptoms in the progression of psychosis. This finding helps clarify previous inconsistent reports and highlights the need for further research into pituitary gland-related factors in individuals with psychosis.