Effects of psilocybin, psychedelic mushroom extract and 5-hydroxytryptophan on brain immediate early gene expression: Interaction with serotonergic receptor modulators.

Background: Immediate early genes (IEGs) are rapidly activated and initiate diverse cellular processes including neuroplasticity. We report the effect of psilocybin (PSIL), PSIL-containing psychedelic mushroom extract (PME) and 5-hydroxytryptophan (5-HTP) on expression of the IEGs, cfos, egr1, and egr2 in mouse somatosensory cortex (SSC). Methods: In our initial experiment, male C57Bl/6j mice were injected with PSIL 4.4 mg/kg or 5-HTP 200 mg/kg, alone or immediately preceded by serotonergic receptor modulators. IEG mRNA expression 1 hour later was determined by real time qPCR. In a replication study a group of mice treated with PME was added. Results: In our initial experiment, PSIL but not 5-HTP significantly increased expression of all three IEGs. No correlation was observed between the head twitch response (HTR) induced by PSIL and its effect on the IEGs. The serotonergic receptor modulators did not significantly alter PSIL-induced IEG expression, with the exception of the 5-HT2C antagonist (RS102221), which significantly enhanced PSIL-induced egr2 expression. 5-HTP did not affect IEG expression. In our replication experiment, PSIL and PME upregulated levels of egr1 and cfos while the upregulation of egr2 was not significant. Conclusions: We have shown that PSIL and PME but not 5-HTP (at a dose sufficient to induce HTR), induced a significant increase in cfos and egr1 expression in mouse SSC. Our findings suggest that egr1 and cfos expression may be associated with psychedelic effects.

Machine learning-enhanced noninvasive prenatal testing of monogenic disorders.

OBJECTIVE: Single-nucleotide variants (SNVs) are of great significance in prenatal diagnosis as they are the leading cause of inherited single-gene disorders (SGDs). Identifying SNVs in a non-invasive prenatal screening (NIPS) scenario is particularly challenging for maternally inherited SNVs. We present an improved method to predict inherited SNVs from maternal or paternal origin in a genome-wide manner. METHODS: We performed SNV-NIPS based on the combination of fragments of cell free DNA (cfDNA) features, Bayesian inference and a machine-learning (ML) prediction refinement step using random forest (RF) classifiers trained on millions of non-pathogenic variants. We next evaluate the real-world performance of our refined method in a clinical setting by testing our models on 16 families with singleton pregnancies and varying fetal fraction (FF) levels, and validate the results over millions of inherited variants in each fetus. RESULTS: The average area under the ROC curve (AUC) values are 0.996 over all families for paternally inherited variants, 0.81 for the challenging maternally inherited variants, 0.86 for homozygous biallelic variants and 0.95 for compound heterozygous variants. Discriminative AUCs were achieved even in families with a low FF. We further investigate the performance of our method in correctly predicting SNVs in coding regions of clinically relevant genes and demonstrate significantly improved AUCs in these regions. Finally, we focus on the pathogenic variants in our cohort and show that our method correctly predicts if the fetus is unaffected or affected in all (10/10, 100%) of the families containing a pathogenic SNV. CONCLUSIONS: Overall, we demonstrate our ability to perform genome-wide NIPS for maternal and homozygous biallelic variants and showcase the utility of our method in a clinical setting.

Combining TIGIT blockage with MDSC inhibition hinders breast cancer bone metastasis by activating anti-tumor immunity.

Bone is the most common site of breast cancer metastasis. Bone metastasis are incurable and are associated with severe morbidity. Utilizing an immunocompetent mouse model of spontaneous breast cancer bone metastasis, we profiled the immune transcriptome of bone metastatic lesions and peripheral bone marrow at distinct metastatic stages, revealing dynamic changes during the metastatic process. We show that crosstalk between granulocytes and T cells is central to shaping an immunosuppressive microenvironment. Specifically, we identified the PD-1 and TIGIT signaling axes and the pro-inflammatory cytokine IL1b as central players in the interactions between granulocytes and T cells. Targeting these pathways in vivo resulted in attenuated bone metastasis and improved survival, by reactivating anti-tumor immunity. Analysis of patient samples revealed that TIGIT and IL1b are prominent in human bone metastasis. Our findings suggest that co-targeting immunosuppressive granulocytes and dysfunctional T cells may be a promising novel therapeutic strategy to inhibit bone metastasis.

A New Intervention for Implementation of Pharmacogenetics in Psychiatry: A Description of the PSY-PGx Clinical Study.

(1) Background Pharmacological treatment for psychiatric disorders has shown to only be effective in about one-third of patients, as it is associated with frequent treatment failure, often because of side effects, and a long process of trial-and-error pharmacotherapy until an effective and tolerable treatment is found. This notion emphasizes the urgency for a personalized medicine approach in psychiatry. (2) Methods This prospective patient- and rater-blinded, randomized, controlled study will investigate the effect of dose-adjustment of antidepressants escitalopram and sertraline or antipsychotics risperidone and aripiprazole according to the latest state-of-the-art international dosing recommendations for CYP2C19 and CYP2D6 metabolizer status in patients with mood, anxiety, and psychotic disorders. A total sample of N = 2500 will be recruited at nine sites in seven countries (expected drop-out rate of 30%). Patients will be randomized to a pharmacogenetic group or a dosing-as-usual group and treated over a 24-week period with four study visits. The primary outcome is personal recovery using the Recovery Assessment Scale as assessed by the patient (RAS-DS), with secondary outcomes including clinical effects (response or symptomatic remission), side effects, general well-being, digital phenotyping, and psychosocial functioning. (3) Conclusions This is, to our knowledge, the first international, multi-center, non-industry-sponsored randomized controlled trial (RCT) that may provide insights into the effectiveness and utility of implementing pharmacogenetic-guided treatment of psychiatric disorders, and as such, results will be incorporated in already available dosing guidelines.

Opioid trends and risk factors for sustained use among children and adolescents in Israel: a retrospective cohort study.

ABSTRACT: Despite growing global concern over opioids, little is known about the epidemiology of opioid use in children and adolescents. This retrospective study investigated opioid use trends and identified risk factors associated with sustained opioid use among outpatient children and adolescents in Israel. Electronic health records of 110,955 children and adolescents were used to establish opioid purchase trends in outpatient settings between 2003 and 2021. Of these, data from 2012 to 2021, n = 32,956, were included in a Cox proportional hazards analysis to identify demographic, clinical, and pharmacological risk factors for sustained opioid use. An increase in opioid use was observed, with a notable rise among strong opioids, peripheral areas, and noncancer patients. Prevalence of sustained opioid users was approximately 2.5%. Risk factors with significant adjusted hazard ratios for sustained use included history of frequent doctor visits 1.82 (95% CI [1.50-2.22]) and drug purchases 1.30 (95% CI [1.07-1.58]), malignancy 1.50 (95% CI [1.07-2.09]), history of cardiovascular (1.44 (95% CI [1.04-1.98]) and pain-related conditions 1.34 (95% CI [1.14-1.58]), and different opioid substances (relative to codeine use): tramadol 2.38 (95% CI [1.73-3.27]), oxycodone 4.29 (95% CI [3.00-6.16]), and "other strong opioids" 6.05 (95% CI [3.59-10.2]). Awareness of observed increase in opioid purchases is crucial for doctors and public health practitioners. Additional monitoring and secondary prevention of children and adolescents possessing the identified risk factors should facilitate where appropriate reducing sustained opioid use when it is unnecessary.

Revealing the tumor suppressive sequence within KL1 domain of the hormone Klotho.

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1-340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1-320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/ß-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and ß sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho's tumor suppressor activity and reveals a critical role in the 320-340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.

Genomic Markers Associated with Cytomegalovirus DNAemia in Kidney Transplant Recipients.

Human cytomegalovirus (CMV) is a major pathogen after solid organ transplantation, leading to high morbidity and mortality. Transplantation from a CMV-seropositive donor to a CMV-seronegative recipient (D+/R-) is associated with high risk of CMV disease. However, that risk is not uniform, suggesting a role for host factors in immune control of CMV. To identify host genetic factors that control CMV DNAemia post transplantation, we performed a whole-exome association study in two cohorts of D+/R- kidney transplant recipients. Quantitative CMV DNA was measured for at least one year following transplantation. Several CMV-protective single-nucleotide polymorphisms (SNPs) were identified in the first cohort (72 patients) but were not reproducible in the second cohort (126 patients). A meta-analysis of both cohorts revealed several SNPs that were significantly associated with protection from CMV DNAemia. The copy number variation of several genes was significantly different between recipients with and without CMV DNAemia. Amongst patients with CMV DNAemia in the second cohort, several variants of interest (p < 5 × 10-5), the most common of which was NLRC5, were associated with peak viral load. We provide new predictive genetic markers for protection of CMV DNAemia. These markers should be validated in larger cohorts.

Unexpected gender differences in progressive supranuclear palsy reveal efficacy for davunetide in women.

Progressive supranuclear palsy (PSP) is a pure tauopathy, implicating davunetide, enhancing Tau-microtubule interaction, as an ideal drug candidate. However, pooling patient data irrespective of sex concluded no efficacy. Here, analyzing sex-dependency in a 52 week-long- PSP clinical trial (involving over 200 patients) demonstrated clear baseline differences in brain ventricular volumes, a secondary endpoint. Dramatic baseline ventricular volume-dependent/volume increase correlations were observed in 52-week-placebo-treated females (r = 0.74, P = 2.36-9), whereas davunetide-treated females (like males) revealed no such effects. Assessment of primary endpoints, by the PSP Rating Scale (PSPRS) and markedly more so by the Schwab and England Activities of Daily Living (SEADL) scale, showed significantly faster deterioration in females, starting at trial week 13 (P = 0.01, and correlating with most other endpoints by week 52). Twice daily davunetide treatments slowed female disease progression and revealed significant protection according to the SEADL scale as early as at 39 weeks (P = 0.008), as well as protection of the bulbar and limb motor domains considered by the PSPRS, including speaking and swallowing difficulties caused by brain damage, and deterioration of fine motor skills, respectably (P = 0.01), at 52 weeks. Furthermore, at 52 weeks of trial, the exploratory Geriatric Depression Scale (GDS) significantly correlated with the SEADL scale deterioration in the female placebo group and demonstrated davunetide-mediated protection of females. Female-specific davunetide-mediated protection of ventricular volume corresponded to clinical efficacy. Together with the significantly slower disease progression seen in men, the results reveal sex-based drug efficacy differences, demonstrating the neuroprotective and disease-modifying impact of davunetide treatment for female PSP patients.

Impaired myelin ultrastructure is reversed by citalopram treatment in a mouse model for major depressive disorder.

Major depressive disorder (MDD) is the most common and widespread mental disorder. Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for MDD. The relation between the inhibition of serotonin reuptake in the central nervous system and remission from MDD remains controversial, as reuptake inhibition occurs rapidly, but remission from MDD takes weeks to months. Myelination-related deficits and white matter abnormalities were shown to be involved in psychiatric disorders such as MDD. This may explain the delay in remission following SSRI administration. The raphe nuclei (RN), located in the brain stem, consist of clusters of serotonergic (5-HT) neurons that project to almost all regions of the brain. Thus, the RN are an intriguing area for research of the potential effect of SSRI on myelination, and their involvement in MDD. MicroRNAs (miRNAs) regulate many biological features that might be altered by antidepressants. Two cohorts of chronic unpredictable stress (CUS) mouse model for depression underwent behavioral tests for evaluating stress, anxiety, and depression levels. Following application of the CUS protocol and treatment with the SSRI, citalopram, 48 mice of the second cohort were tested via magnetic resonance imaging and diffusion tensor imaging for differences in brain white matter tracts. RN and superior colliculus were excised from both cohorts and measured for changes in miRNAs, mRNA, and protein levels of candidate genes. Using MRI-DTI scans we found lower fractional anisotropy and axial diffusivity in brains of stressed mice. Moreover, both miR-30b-5p and miR-101a-3p were found to be downregulated in the RN following CUS, and upregulated following CUS and citalopram treatment. The direct binding of these miRNAs to Qki, and the subsequent effects on mRNA and protein levels of myelin basic protein (Mbp), indicated involvement of these miRNAs in myelination ultrastructure processes in the RN, in response to CUS followed by SSRI treatment. We suggest that SSRIs are implicated in repairing myelin deficits resulting from chronic stress that leads to depression.

mRNA splicing is modulated by intronic microRNAs.

Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high-affinity binding, namely more than eight base pairs, with the 5'SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5'SS base-pairing or other regulatory obstructions. Here, we show in vitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5'SS, and hence promotes the inclusion of exons containing 5'SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing.

Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4.

Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole-exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2-causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/ß-catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/ß-catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain-of-function effect.

Can RNA Affect Memory Modulation? Implications for PTSD Understanding and Treatment.

Memories are a central aspect of our lives, but the mechanisms underlying their formation, consolidation, retrieval, and extinction remain poorly understood. In this review, we explore the molecular mechanisms of memory modulation and investigate the effects of RNA on these processes. Specifically, we examine the effects of time and location on gene expression alterations. We then discuss the potential for harnessing these alterations to modulate memories, particularly fear memories, to alleviate post-traumatic stress disorder (PTSD) symptoms. The current state of research suggests that transcriptional changes play a major role in memory modulation and targeting them through microRNAs may hold promise as a novel approach for treating memory-related disorders such as PTSD.

Hippocampal differential expression underlying the neuroprotective effect of delta-9-tetrahydrocannabinol microdose on old mice.

Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound of the cannabis plant and an exogenous ligand of the endocannabinoid system. In previous studies, we demonstrated that a single microdose of THC (0.002 mg/kg, 3-4 orders of magnitude lower than the standard dose for rodents) exerts distinct, long-term neuroprotection in model mice subjected to acute neurological insults. When administered to old, healthy mice, the THC microdose induced remarkable long-lasting (weeks) improvement in a wide range of cognitive functions, including significant morphological and biochemical brain alterations. To elucidate the mechanisms underlying these effects, we analyzed the gene expression of hippocampal samples from the model mice. Samples taken 5 days after THC treatment showed significant differential expression of genes associated with neurogenesis and brain development. In samples taken 5 weeks after treatment, the transcriptional signature was shifted to that of neuronal differentiation and survival. This study demonstrated the use of hippocampal transcriptome profiling in uncovering the molecular basis of the atypical, anti-aging effects of THC microdose treatment in old mice.

A novel SLC25A13 gene splice site variant causes Citrin deficiency in an infant.

Citrin deficiency is an autosomal recessive disorder associated with SLC25A13 gene pathogenic variants, with more than a hundred known at present. It manifests in neonates as failure to thrive and acute liver insufficiency. We herein describe a case of a 4-week-old infant who presented with insufficient weight gain and liver failure accompanied by hyperammonemia. She was diagnosed with Citrin deficiency after a thorough biochemical and molecular analysis including amino acid profile, DNA sequencing of genes of interest and RNA splice site evaluation, to reveal a yet unknown damaging variant of the SLC25A13 gene.

Deoxyhypusine hydroxylase: A novel therapeutic target differentially expressed in short-term vs long-term survivors of glioblastoma.

Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long-term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA-seq and mass spectrometry (MS)-based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer-related pathways and some less established that showed higher expression in short-term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusual amino acid essential for the function of the eukaryotic translation initiation factor 5A (eIF5A), which promotes tumor growth. We consequently validated DOHH overexpression in STS samples by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. We further showed robust inhibition of proliferation, migration and invasion of GB cells following silencing of DOHH with short hairpin RNA (shRNA) or inhibition of its activity with small molecules, ciclopirox and deferiprone. Moreover, DOHH silencing led to significant inhibition of tumor progression and prolonged survival in GB mouse models. Searching for a potential mechanism by which DOHH promotes tumor aggressiveness, we found that it supports the transition of GB cells to a more invasive phenotype via epithelial-mesenchymal transition (EMT)-related pathways.

Imputation of single-cell transcriptome data enables the reconstruction of networks predictive of breast cancer metastasis.

Single-cell transcriptome data provide a unique opportunity to explore the gene networks of a particular cell type. However, insufficient capture rate and high dimensionality of single-cell RNA sequencing (scRNA-seq) data challenge cell-type-specific gene network (CGN) reconstruction. Here, we demonstrated that the imputation of scRNA-seq data enables reconstruction of CGNs by effective retrieval of gene functional associations. We reconstructed CGNs for seven primary and nine metastatic breast cancer cell lines using scRNA-seq data with imputation. Key genes for primary or metastatic cell lines were prioritized based on network centrality measures and CGN hub genes that were presumed to be the major determinant of cell type characteristics. To identify novel genes in breast cancer metastasis, we used the average rank difference of centrality between the primary and metastatic cell lines. Genes predicted using CGN centrality analysis were more enriched for known breast cancer metastatic genes than those predicted using differential expression. The molecular chaperone CCT2 was identified as a novel gene for breast metastasis during knockdown assays of several candidate genes. Overall, our study demonstrated an effective CGN reconstruction technique with imputation of scRNA-seq data and the feasibility of identifying key genes for particular cell subsets using single-cell network analysis.

MYC Induces Immunotherapy and IFNγ Resistance Through Downregulation of JAK2.

Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2.

Immunosuppression as a Hub for SARS-CoV-2 Mutational Drift.

The clinical course of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is largely determined by host factors, with a wide range of outcomes. Despite an extensive vaccination campaign and high rates of infection worldwide, the pandemic persists, adapting to overcome antiviral immunity acquired through prior exposure. The source of many such major adaptations is variants of concern (VOCs), novel SARS-CoV-2 variants produced by extraordinary evolutionary leaps whose origins remain mostly unknown. In this study, we tested the influence of factors on the evolutionary course of SARS-CoV-2. Electronic health records of individuals infected with SARS-CoV-2 were paired to viral whole-genome sequences to assess the effects of host clinical parameters and immunity on the intra-host evolution of SARS-CoV-2. We found slight, albeit significant, differences in SARS-CoV-2 intra-host diversity, which depended on host parameters such as vaccination status and smoking. Only one viral genome had significant alterations as a result of host parameters; it was found in an immunocompromised, chronically infected woman in her 70s. We highlight the unusual viral genome obtained from this woman, which had an accelerated mutational rate and an excess of rare mutations, including near-complete truncating of the accessory protein ORF3a. Our findings suggest that the evolutionary capacity of SARS-CoV-2 during acute infection is limited and mostly unaffected by host characteristics. Significant viral evolution is seemingly exclusive to a small subset of COVID-19 cases, which typically prolong infections in immunocompromised patients. In these rare cases, SARS-CoV-2 genomes accumulate many impactful and potentially adaptive mutations; however, the transmissibility of such viruses remains unclear.

Comprehensive Genetic Analysis of Druze Provides Insights into Carrier Screening.

BACKGROUND: Druze individuals, like many genetically homogeneous and isolated populations, harbor recurring pathogenic variants (PV) in autosomal recessive (AR) disorders. METHODS: Variant calling of whole-genome sequencing (WGS) of 40 Druze from the Human Genome Diversity Project (HGDP) was performed (HGDP-cohort). Additionally, we performed whole exome sequencing (WES) of 118 Druze individuals: 38 trios and 2 couples, representing geographically distinct clans (WES-cohort). Rates of validated PV were compared with rates in worldwide and Middle Eastern populations, from the gnomAD and dbSNP datasets. RESULTS: Overall, 34 PVs were identified: 30 PVs in genes underlying AR disorders, 3 additional PVs were associated with autosomal dominant (AD) disorders, and 1 PV with X-linked-dominant inherited disorder in the WES cohort. CONCLUSIONS: The newly identified PVs associated with AR conditions should be considered for incorporation into prenatal-screening options offered to Druze individuals after an extension and validation of the results in a larger study.