The abundantly expressed calcium/calmodulin-dependent protein kinase II (CAMK2), alpha (CAMK2A), and beta (CAMK2B) isoforms are essential for learning and memory formation. Recently, a de novo candidate mutation (p.Arg292Pro) in the gamma isoform of CAMK2 (CAMK2G) was identified in a patient with severe intellectual disability (ID), but the mechanism(s) by which this mutation causes ID is unknown. Here, we identified a second, unrelated individual, with a de novo CAMK2G p.Arg292Pro mutation, and used in vivo and in vitro assays to assess the impact of this mutation on CAMK2G and neuronal function. We found that knockdown of CAMK2G results in inappropriate precocious neuronal maturation. We further found that the CAMK2G p.Arg292Pro mutation acts as a highly pathogenic gain-of-function mutation, leading to increased phosphotransferase activity and impaired neuronal maturation as well as impaired targeting of the nuclear CAMK2G isoform. Silencing the catalytic site of the CAMK2G p.Arg292Pro protein reversed the pathogenic effect of the p.Arg292Pro mutation on neuronal maturation, without rescuing its nuclear targeting. Taken together, our results reveal an indispensable function of CAMK2G in neurodevelopment and indicate that the CAMK2G p.Arg292Pro protein acts as a pathogenic gain-of-function mutation, through constitutive activity toward cytosolic targets, rather than impaired targeting to the nucleus.

A combined in silico method was developed to predict potential protein targets that are involved in cardiotoxicity induced by aconitine alkaloids and to study the quantitative structure⁻toxicity relationship (QSTR) of these compounds. For the prediction research, a Protein-Protein Interaction (PPI) network was built from the extraction of useful information about protein interactions connected with aconitine cardiotoxicity, based on nearly a decade of literature and the STRING database. The software Cytoscape and the PharmMapper server were utilized to screen for essential proteins in the constructed network. The Calcium-Calmodulin-Dependent Protein Kinase II alpha (CAMK2A) and gamma (CAMK2G) were identified as potential targets. To obtain a deeper insight on the relationship between the toxicity and the structure of aconitine alkaloids, the present study utilized QSAR models built in Sybyl software that possess internal robustness and external high predictions. The molecular dynamics simulation carried out here have demonstrated that aconitine alkaloids possess binding stability for the receptor CAMK2G. In conclusion, this comprehensive method will serve as a tool for following a structural modification of the aconitine alkaloids and lead to a better insight into the cardiotoxicity induced by the compounds that have similar structures to its derivatives.

BACKGROUND

Molecular mechanisms underlying psychological sequelae of exposure to stressful experiences, such as posttraumatic stress disorder (PTSD) and depression, are not well understood.

METHODS

Using convergent evidence from animal and human transcriptomic and genomic studies, we aimed to identify genetic mechanisms underlying depression and anxiety after traumatic experiences.

RESULTS

From a transcriptome-wide analysis in mice, we found the Ppm1f gene to be differentially expressed in the amygdala and medial prefrontal cortex (mPFC) a week after immobilization stress. Next, we found that PPM1F messenger RNA levels in human blood were downregulated in cases with symptoms of comorbid PTSD and depression and consistently in cases with anxiety symptoms in a separate human dataset. Furthermore, we showed that a genetic variant of PPM1F, rs17759843, was associated with comorbid PTSD and depression and with PPM1F expression in both human brain and blood. Given prior reported mechanistic links between PPM1F and CAMK2 (CAMKII), we examined blood messenger RNA level of CAMK2G in humans and found it to be lower in cases with comorbid PTSD and depression. We also found that PPM1F protein levels and colocalization with CAMK2G were altered in amygdala and mPFC of male mice. Additionally, we found that a systemic dose of corticosterone blocked the depressive-like phenotype elicited by stress in female mice. Lastly, corticosterone rescued the anxiety-like phenotype and messenger RNA levels of Ppm1f in amygdala and mPFC in male mice and in mPFC of female mice.

CONCLUSIONS

Taken together, our data suggest a mechanistic pathway involving PPM1F and CAMK2G in stress- and trauma-related manifestation of anxiety and depression across species.

CTG repeat expansion (CTG_exp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTG_exp repeat using CRISPR/Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1 induced pluripotent stem cells (iPSC). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR/Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular-signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G) and other DM1-related genes (i.e. NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3 and CRTC2 further confirmed correction of the spliceopathy following CTG_exp repeat excision. The present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTG_exp repeat using CRISPR/Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

Excitation-contraction coupling in normal cardiac function is performed with well balanced and coordinated functioning but with complex dynamic interactions between functionally connected membrane ionic currents. However, their genomic investigations provide essential information on the regulation of diseases by their transcripts. Therefore, we examined the gene expression levels of the most important voltage-gated ionic channels such as Na⁺-channels (SCN5A), Ca²⁺-channels (CACNA1C and CACNA1H), and K⁺-channels, including transient outward (KCND2, KCNA2, KCNA5, KCNA8), inward rectifier (KCNJ2, KCNJ12, KCNJ4), and delayed rectifier (KCNB1) in left ventricular tissues from either ischemic or dilated cardiomyopathy (ICM or DCM). We also examined the mRNA levels of ATP-dependent K⁺-channels (KCNJ11, ABCC9) and ERG-family channels (KCNH2). We further determined the mRNA levels of ryanodine receptors (RyR2; ARVC2), phospholamban (PLB or PLN), SR Ca²⁺-pump (SERCA2; ATP2A1), an accessory protein FKBP12 (PPIASE), protein kinase A (PPNAD4), and Ca²⁺/calmodulin-dependent protein kinase II (CAMK2G). The mRNA levels of SCN5A, CACNA1C, and CACNA1H in both groups decreased markedly in the heart samples with similar significance, while KvLQT1 genes were high with depressed Kv4.2. The KCNJ11 and KCNJ12 in both groups were depressed, while the KCNJ4 level was significantly high. More importantly, the KCNA5 gene was downregulated only in the ICM, while the KCNJ2 was upregulated only in the DCM. Besides, mRNA levels of ARVC2 and PLB were significantly high compared to the controls, whereas others (ATP2A1, PPIASE, PPNAD4, and CAMK2G) were decreased. Importantly, the increases of KCNB1 and KCNJ11 were more prominent in the ICM than DCM, while the decreases in ATP2A1 and FKBP1A were more prominent in DCM compared to ICM. Overall, this study was the first to demonstrate that the different levels of changes in gene profiles via different types of cardiomyopathy are prominent particularly in some K⁺-channels, which provide further information about our knowledge of how remodeling processes can be differentiated in HF originated from different pathological conditions.

RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.

Epilepsy is a complex neurological disorder and a significant health problem. The pathogenesis of epilepsy remains obscure in a significant number of patients and the current treatment options are not adequate in about a third of individuals which were known as refractory epilepsies (RE). Network medicine provides an effective approach for studying the molecular mechanisms underlying complex diseases. Here we integrated 1876 disease-gene associations of RE and located those genes to human protein-protein interaction (PPI) network to obtain 42 significant RE-associated disease modules. The functional analysis of these disease modules showed novel molecular pathological mechanisms of RE, such as the novel enriched pathways (e.g., "presynaptic nicotinic acetylcholine receptors", "signaling by insulin receptor"). Further analysis on the relationships between current drug targets and the RE-related disease genes showed the rational mechanisms of most antiepileptic drugs. In addition, we detected ten potential novel drug targets (e.g., KCNA1, KCNA4-6, KCNC3, KCND2, KCNMA1, CAMK2G, CACNB4 and GRM1) located in three RE related disease modules, which might provide novel insights into the new drug discovery for RE therapy.

Background: Coronary artery disease (CAD) is a type of cardiovascular disease that greatly hurts the health of human beings. Diabetic status is one of the largest clinical factors affecting CAD-associated gene expression changes. Most of the studies focus on diabetic patients, whereas few have been done for non-diabetic patients. Since the pathophysiological processes may vary among these patients, we cannot simply follow the standard based on the data from diabetic patients. Therefore, the prognostic and predictive diagnostic biomarkers for CAD in non-diabetic patient need to be fully recognized.

Materials and methods: To screen out candidate genes associated with CAD in non-diabetic patients, weighted gene co-expression network analysis (WGCNA) was constructed to conduct an analysis of microarray expression profiling in patients with CAD. First, the microarray data GSE20680 and GSE20681 were downloaded from NCBI. We constructed co-expression modules via WGCNA after excluding the diabetic patients. As a result, 18 co-expression modules were screened out, including 1,225 differentially expressed genes (DEGs) that were obtained from 152 patients (luminal stenosis ≥50% in at least one major vessel) and 170 patients (stenosis of <50%). Subsequently, a Pearson's correlation analysis was conducted between the modules and clinical traits. Then, a functional enrichment analysis was conducted, and we used gene network analysis to reveal hub genes. Last, we validated the hub genes with peripheral blood samples in an independent patient cohort using RT-qPCR.

Results: The results showed that the midnight blue module and the yellow module played vital roles in the pathogenesis of CAD in non-diabetic patients. Additionally, CD40, F11R, TNRC18, and calcium/calmodulin-dependent protein kinase type II gamma (CAMK2G) were screened out and validated using enzyme-linked immunosorbent assay (ELISA) in an independent patient cohort and immunohistochemical (IHC) staining in an atherosclerosis mouse model.

Conclusion: Our findings demonstrate that hub genes, CD40, F11R, TNRC18, and CAMK2G, are surrogate diagnostic biomarkers and/or therapeutic targets for CAD in non-diabetic patients and require deeper validation.

Keywords: biomarkers; coronary artery disease; function enrichment analysis; non-diabetic patients; weighted gene co-expression network analysis.

Background: Litter size is an important index of mammalian prolificacy and is determined by the ovulation rate. The ovary is a crucial organ for mammalian reproduction and is associated with follicular development, maturation and ovulation. However, prolificacy is influenced by multiple factors, and its molecular regulation in the follicular phase remains unclear.

Methods: Ten female goats with no significant differences in age and weight were randomly selected and divided into either the high-yielding group (n = 5, HF) or the low-yielding group (n = 5, LF). Ovarian tissues were collected from goats in the follicular phase and used to construct mRNA and miRNA sequencing libraries to analyze transcriptomic variation between high- and low-yield Yunshang black goats. Furthermore, integrated analysis of the differentially expressed (DE) miRNA-mRNA pairs was performed based on their correlation. The STRING database was used to construct a PPI network of the DEGs. RT-qPCR was used to validate the results of the predicted miRNA-mRNA pairs. Luciferase analysis and CCK-8 assay were used to detect the function of the miRNA-mRNA pairs and the proliferation of goat granulosa cells (GCs).

Results: A total of 43,779 known transcripts, 23,067 novel transcripts, 424 known miRNAs and 656 novel miRNAs were identified by RNA-seq in the ovaries from both groups. Through correlation analysis of the miRNA and mRNA expression profiles, 263 negatively correlated miRNA-mRNA pairs were identified in the LF vs. HF comparison. Annotation analysis of the DE miRNA-mRNA pairs identified targets related to biological processes such as "estrogen receptor binding (GO:0030331)", "oogenesis (GO:0048477)", "ovulation cycle process (GO:0022602)" and "ovarian follicle development (GO:0001541)". Subsequently, five KEGG pathways (oocyte meiosis, progesterone-mediated oocyte maturation, GnRH signaling pathway, Notch signaling pathway and TGF-β signaling pathway) were identified in the interaction network related to follicular development, and a PPI network was also constructed. In the network, we found that CDK12, FAM91A1, PGS1, SERTM1, SPAG5, SYNE1, TMEM14A, WNT4, and CAMK2G were the key nodes, all of which were targets of the DE miRNAs. The PPI analysis showed that there was a clear interaction among the CAMK2G, SERTM1, TMEM14A, CDK12, SYNE1 and WNT4 genes. In addition, dual luciferase reporter and CCK-8 assays confirmed that miR-1271-3p suppressed the proliferation of GCs by inhibiting the expression of TXLNA.

Conclusions: These results increase the understanding of the molecular mechanisms underlying goat prolificacy. These results also provide a basis for studying interactions between genes and miRNAs, as well as the functions of the pathways in ovarian tissues involved in goat prolificacy in the follicular phase.

Keywords: Follicular phase; Ovarian tissues; Prolific goats; miRNA-mRNA pairs.

Although dramatic progress has been achieved in the understanding and treatment of multiple sclerosis (MS) and ischemic stroke (IS), more precise and instructive support is required for further research. Recent large-scale genome-wide association studies (GWASs) have already revealed risk variants for IS and MS, but the common genetic etiology between MS and IS remains an unresolved issue. This research was designed to overlapping genes between MS and IS and unmask their transcriptional features. We designed a three-section analysis process. Firstly, we computed gene-based analyses of MS GWAS and IS GWAS data sets by VGEAS2. Secondly, overlapping genes of significance were identified in a meta-analysis using the Fisher's procedure. Finally, we performed gene expression analyses to confirm transcriptional changes. We identified 24 shared genes with Bonferroni correction (P _combined < 2.31E-04), and five (FOXP1, CAMK2G, CLEC2D, LBH, and SLC2A4RG) had significant expression differences in MS and IS gene expression omnibus data sets. These meaningful shared genes between IS and MS shed light on the underlying genetic etiologies shared by the diseases. Our results provide a basis for in-depth genomic studies of associations between MS and IS.

Keywords: gene expression analyses; gene-based test; genome-wide association studies; ischemic stroke; multiple sclerosis.

Background & aims: Maternal folic acid (FA) supplement (FolS) programs the early development of an offspring. The onset of complex diseases at a later stage of life has been evidently linked with maternal FA ingestion. However, little is known regarding the underlying molecule fingerprints of the offspring. Here, we analyze the influence of maternal FolS on the metabolism of the adult offspring rats using the integrated metabolomics-proteomics.

Methods: Twenty pregnant female rats were randomly assigned to a FA supplement (FolS group) or control group which were fed AIN93G diet with 2 or 5 mg/kg FA, respectively. The blood samples from the offspring at 0, 3 and 7 weeks after birth were collected. The brain samples were obtained from the offspring at 7 weeks after birth. Serum and brain metabolite profiles were performed by UPLC-MS/MS and the brain proteomics analysis was obtained using iTRAQ-based quantitative proteomics.

Results: The metabolic change of the offspring for the maternal FA supplement is characterized by the phospholipids, fatty acid and amino acids, which are involved in linoleic acid, docosahexaenoic acid, glycerophosphocholine, lysophosphatidylcholine, tryptophan, glycine, arachidonic acid, γ-aminobutyric acid, and so on. Using iTRAQ-based quantitative proteomics analysis, 51 differential proteins in the brain are identified, which provides valuable insight into the underlying mechanisms of the offspring after the maternal FolS. These results demonstrate neural development related metabolites and proteins, such as docosahexaenoic acid, glycine, tryptophan, γ-aminobutyric acid, dopaminergic synapse related proteins including G protein, PPP1R1B and CAMK2G, are significantly altered, which suggests that the active neural conduction occurs in the offspring after maternal FA supplement. The behavioral testing demonstrates that the high level of memory is observed in rats with FA supplement.

Conclusions: We conceive that the alterations of metabolites and protein in the offspring are associated with the maternal FA supplement and these alterations are involved in the neural development, although such animal data are limited in their ability to mimic metabolic outcomes in humans.

Keywords: Folic acid; Gestational nutrition supplement; Metabolomics; Neural development; Proteomics.

Testosterone can induce impulsivity, a behavioral impairment associated with various psychiatric illnesses. The molecular mechanisms associated with testosterone-induced impulsivity are unclear. Our earlier studies showed that supraphysiological doses of testosterone to rats induced impulsive behavior, impacted hypothalamic-pituitary-adrenal axis (HPA) and hypothalamic-pituitary-gonadal axis interactions, and altered α_2A adrenergic receptors in prefrontal cortex (PFC). Owing to the importance of GABAergic system in impulsivity and memory, the present study examines whether testosterone-mediated impulsivity is associated with changes in the expression of Gamma-Aminobutyric Acid (GABA) A and B receptor subunit transcripts (Gabra1, Gabra2, Gabra2 transcript variant 2, Gabra3, Gabra4, Gabra5, Gabra6, Gabrb1, Gabrb2, Gabrb3, Gabrg1, Gabrg2, Gabrg3, Gabbr1, Gabbr2) in rat PFC, and whether testosterone influences GABA_A receptor subunit organization. We studied GABA receptor functions by examining GABA receptor-mediated calcium/calmodulin-dependent kinase signaling genes (Calm1, Calm2, Calm3, Camk2a, Camk2b, Camk2g, Camk2d, Camk4) in the testosterone-induced impulsivity model. Rats were left untreated as controls (C), gonadectomized (GDX), or GDX and injected with supraphysiological doses of testosterone (T). Impulsive behavior was examined using the go/no-go paradigm. Gene expression was studied using qRT-PCR and GABA_A subunit reorganization using cross correlation. Our findings show that expressions of select GABA_A receptor subunits (Gabra3, Gabra5, Gabra6) were significantly upregulated in PFC of T group compared to GDX or C groups. GABA_A receptor subunit organization was different in C, T, and GDX groups. Additionally, Camk4 expression was significantly downregulated in T compared to C group. Our findings suggest that specific GABA_A receptor subunit expression, their reorganization, and Camk4-mediated functions may be associated with testosterone-mediated impulsivity.

Keywords: GABAA receptor; impulsivity; rodent model; testosterone; transcript level.

Fluvoxamine, a common antidepressant agent, is designed to exert its pharmacological effect by inhibiting synaptic serotonin reuptake. However, increasing evidence has demonstrated that σ1 receptors are likely to be involved in the mechanism of action of fluvoxamine. The present study aimed to observe the effects of fluvoxamine on the expression levels of mammalian target of rapamycin (mTOR), Ca2+/calmodulin-dependent protein kinase 2γ (Camk2γ) and glycogen synthase kinase-3β (GSK-3β) in fluvoxamine-treated N2a cells and attempted to elucidate whether σ1 receptors mediate the pharmacological effects of fluvoxamine. The N2a cells were randomly divided into three groups (each n=6): DMEM group (D group), 0.5 μmol/l fluvoxamine group (F group) and 0.2 μmol/l BD1047 (a σ1 receptor antagonist) + 0.5 μmol/l fluvoxamine group (BF group). Western blotting was used to determine the expression levels of mTOR, Camk2γ and GSK-3β in the cultured N2a cells after two days of incubation. The F group exhibited significant increases in the expression levels of mTOR and Camk2γ and a significant reduction in the expression levels of GSK-3β compared with those in the D group (P<0.01). By contrast, the BF group demonstrated significant reductions in the expression levels of mTOR and Camk2γ and a significant increase in the expression levels of GSK-3β, compared with those in the F group (P<0.01). These results suggest that σ1 receptors mediate fluvoxamine-elicited changes in the expression levels of mTOR, Camk2γ and GSK-3β in N2a cells, which indicates that σ1 receptors are likely to be involved in the pharmacological effects of fluvoxamine.

Multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is a multi-gene family with isoform-specific regulation of vascular smooth muscle (VSM) functions. In previous studies we found that vascular injury resulted in VSM de-differentiation and reduced expression of the CaMKIIg isoform in medial wall VSM. Smooth muscle knockout of CaMKIIg enhanced injury-induced VSM neointimal hyperplasia whereas CaMKIIg over-expression inhibited VSM proliferation and neointimal formation. In this study, we evaluated DNA cytosine methylation/demethylation as a mechanism for regulating CaMKII isoform expression in VSM. Inhibition of cytosine methylation with 5-Aza-2'-deoxycytidine, significantly up-regulated CaMKIIg expression in cultured VSM cells and inhibited CaMKIIg downregulation in organ-cultured aorta ex vivo. Using methylated cytosine immuno-precipitation, the rat Camk2g promoter was found hypomethylated in differentiated VSM, whereas injury- or cell culture-induced VSM de-differentiation coincided with Camk2g promoter methylation and decreased expression. We report for the first time, that VSM cell phenotype switching is accompanied by marked induction of thymine DNA glycosylase (TDG) protein and mRNA expression in injured arteries in vivo and in cultured VSM synthetic phenotype cells. Silencing Tdg in VSM promoted expression of CaMKIIg and differentiation markers, including myocardin, and inhibited VSM cell proliferation and injury-induced neointima formation. This study indicates that CaMKIIg expression in VSM is regulated by cytosine methylation/demethylation and that TDG is an important determinant of this process and, more broadly, VSM phenotype switching and function.

The coexistence of coronary artery disease (CAD) and chronic kidney disease (CKD) implies overlapped genetic foundation. However, the common genetic determination between the two diseases remains largely unknown. Relying on summary statistics publicly available from large scale genome-wide association studies (n = 184,305 for CAD and n = 567,460 for CKD), we observed significant positive genetic correlation between CAD and CKD (r _g = 0.173, p = 0.024) via the linkage disequilibrium score regression. Next, we implemented gene-based association analysis for each disease through MAGMA (Multi-marker Analysis of GenoMic Annotation) and detected 763 and 827 genes associated with CAD or CKD (FDR < 0.05). Among those 72 genes were shared between the two diseases. Furthermore, by integrating the overlapped genetic information between CAD and CKD, we implemented two pleiotropy-informed informatics approaches including cFDR (conditional false discovery rate) and GPA (Genetic analysis incorporating Pleiotropy and Annotation), and identified 169 and 504 shared genes (FDR < 0.05), of which 121 genes were simultaneously discovered by cFDR and GPA. Importantly, we found 11 potentially new pleiotropic genes related to both CAD and CKD (i.e., ARHGEF19, RSG1, NDST2, CAMK2G, VCL, LRP10, RBM23, USP10, WNT9B, GOSR2, and RPRML). Five of the newly identified pleiotropic genes were further repeated via an additional dataset CAD available from UK Biobank. Our functional enrichment analysis showed that those pleiotropic genes were enriched in diverse relevant pathway processes including quaternary ammonium group transmembrane transporter, dopamine transport. Overall, this study identifies common genetic architectures overlapped between CAD and CKD and will help to advance understanding of the molecular mechanisms underlying the comorbidity of the two diseases.

Keywords: chronic kidney disease; coronary artery disease; gene-based association analysis; genome-wide association study; pleiotropic gene; pleiotropy-informed integrative analysis.

Persistent drug-seeking behavior has been associated with deficits in neural circuits that regulate the extinction of addictive behaviors. Although there is extensive data that associates addiction phases with neuroplasticity changes in the reward circuit, little is known about the underlying mechanisms of extinction learning of opioid associated cues. Here, we combined morphine-conditioned place preference (CPP) with real-time polymerase chain reaction (RT-PCR) to identify the effects of extinction training on the expression of genes (mRNAs) associated with synaptic plasticity and opioid receptors in the ventral striatum/nucleus accumbens (VS/NAc). Following morphine extinction training, we identified two animal subgroups showing either extinction (low CPP) or extinction-resistance (high CPP). A third group were conditioned to morphine but did not receive extinction training (sham-extinction; high CPP). RT-PCR results showed that brain derived neurotrophic factor (Bdnf) was upregulated in rats showing successful extinction. Conversely, the lack of extinction training (sham-extinction) upregulated genes associated with kinases (Camk2g), neurotrophins (Ngfr), synaptic connectivity factors (Ephb2), glutamate neurotransmission (Grm8) and opioid receptors (μ1, Δ1). To further identify genes modulated by morphine itself, comparisons with their saline-counterparts were performed. Results revealed that Bdnf was consistently upregulated in the extinction group. Alternatively, widespread gene modulation was observed in the group with lack of extinction training (i.e. Drd2, Cnr1, Creb, μ1, Δ1) and the group showing extinction resistance (i.e. Crem, Rheb, Tnfa). Together, our study builds on the identification of putative genetic markers for the extinction learning of drug-associated cues.

CaMKIIγ, the predominant CaMKII isoform in mouse eggs, controls egg activation by regulating cell cycle resumption. In this study we further characterize the involvement and specificity of CaMKIIγ in mouse egg activation. Using exogenous expression of different cRNAs in Camk2g(-/-) eggs, we show that the other multifunctional CaM kinases, CaMKI, and CaMKIV, are not capable of substituting CaMKIIγ to initiate cell cycle resumption in response to a rise in intracellular Ca (2+). Exogenous expression of Camk2g or Camk2d results in activation of nearly 80% of Camk2g(-/-) MII eggs after stimulation with SrCl 2, which does not differ from the incidence of activation of wild-type eggs expressing exogenous Egfp. In contrast, none of the Camk2g(-/-) MII eggs expressing Camk1 or Camk4 activate in response to SrCl 2 treatment. Expression of a constitutively active form of Camk4 (ca-Camk4), but not Camk1, triggers egg activation. EMI2, an APC/C repressor, is a key component in regulating egg activation downstream of CaMKII in both Xenopus laevis and mouse. We show that exogenous expression of either Camk2g, Camk2d, or ca-Camk4, but not Camk1, Camk4, or a catalytically inactive mutant form of CaMKIIγ (kinase-dead) in Camk2g(-/-) mouse eggs leads to almost complete degradation (~90%) of exogenously expressed EMI2 followed by cell cycle resumption. Thus, degradation of EMI2 following its phosphorylation specifically by CaMKII is mechanistically linked to and promotes cell cycle resumption in MII eggs.

Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation-contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart's calcium signaling and transcriptional response to hypoxia.

Annexin A4 (ANXA4) is a Ca²⁺- and phospholipid-binding protein that belongs to the annexin family, which is involved in the development of multiple tumour types via NF-κB signalling. In this study, we verified the high expression and membrane-cytoplasm translocation of ANXA4 in colorectal carcinoma (CRC). Calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) was found to be important for high ANXA4 expression in CRC, whereas carbonic anhydrase (CA1) promoted ANXA4 aggregation in the cell membrane. An increased Ca²⁺ concentration attenuated the small ubiquitin-like modifier (SUMO) modification of cytoplasmic ANXA4 and ANXA4 stabilization, and relatively high expression of ANXA4 promoted CRC tumorigenesis and epithelial-mesenchymal transition (EMT).

Keywords: SUMO; annexin A4; calcium/calmodulin-dependent protein kinase II gamma; carbonic anhydrase; membrane-cytoplasm translocation.

Introduction: Prenatal stress (PS) can cause depression in offspring. However, the underlying biological mechanism of these influences is still unclear. This work was implemented to investigate the molecular mechanisms of depressive-like behavior of offspring rats insulted with PS.

Methods: Relative quantitative phosphoproteomics of the hippocampus of PS susceptibility (PS-S) and control (CON) rat offspring was performed using liquid chromatography-tandem mass spectrometry to confirm known pathways and to identify new mechanisms involved in depression.

Results: A total of 6790 phosphopeptides, 9817 phosphorylation sites, and 2978 phosphoproteins were detected. Among the 2978 phosphoproteins, 1760 (59.09%) had more than two phosphorylated sites, the ENSRNOP00000023460 protein had more than 117 phosphorylated sites, and the average distribution of modification sites per 100 amino acids was 2.97. There were 197 different phosphopeptides, including 140 increased phosphopeptides and 57 decreased phosphopeptides in the PS-S offspring rats, compared to the CON offspring rats. These differential phosphopeptides corresponded to 100 upregulated and 44 downregulated phosphoproteins, respectively. Gene ontology enrichment analysis revealed that these different phosphoproteins in the top five enriched terms in the cellular component, molecular function, and biological proces categories were involved in a total of 35 different phosphoproteins, and these phosphoproteins were mainly related to myelin-, microtubule- and synapse-associated proteins. The enrichment of Kyoto Encyclopedia of Genes and Genome pathways was found to be involved in many essential biological pathways, and the top five pathways included amphetamine addiction, insulin secretion, Cushing syndrome, and the circadian entrainment signaling pathway. These first five pathways were related to nine phosphoproteins, including Adcy9, Apc, Cacna1c, Camk2a, Camk2b, Camk2g, Ctnnd2, Grin2a, and Stx1a. The full data are available via ProteomeXchange with identifier PXD019117.

Conclusion: We preliminarily identified 144 different phosphoproteins involved in myelin, microtubule, and synapse formation and plasticity in the hippocampus of susceptible offspring rats exposed to PS.

Keywords: depressive-like behaviors; hippocampus; offspring; phosphoproteomics; prenatal stress.

Although JAK1/2 inhibition is effective into alleviating symptoms of myelofibrosis (MF), it does not result in the eradication of MF clones, which can lead to inhibitor-resistant clones emerging during the treatment. Here we established iPS cells derived from MF patient samples (MF-iPSCs) harboring JAK2 V617F, CALR type 1, or CALR type 2 mutations. We demonstrated that these cells faithfully recapitulate the drug sensitivity of the disease. These cells were utilized for chemical screening and calcium/calmodulin-dependent protein kinase 2 (CAMK2) was identified as a promising therapeutic target. MF model cells and mice induced by MPL W515L, another type of mutations recurrently detected in MF patients were used to elucidate the therapeutic potential of CAMK2 inhibition. CAMK2 inhibition was effective against JAK2 inhibitor-sensitive and JAK2 inhibitor-resistant cells. Further research revealed CAMK2 gamma subtype was important in MF model cells induced by MPL W515L. We showed that CAMK2G hetero knockout in the primary bone marrow cells expressing MPL W515Ldecreased colony-forming capacity. CAMK2G inhibition with berbamine, a CAMK2G inhibitor, significantly prolonged survival and reduced disease phenotypes such as splenomegaly and leukocytosis in a MF mouse model induced by MPL W515L. We investigated the molecular mechanisms underlying the therapeutic effect of CAMK2G inhibition and found that CAMK2G is activated by MPL signaling in MF model cells and is an effector in the MPL-JAK2 signaling pathway in these cells. These results indicate CAMK2G plays an important role in MF, and CAMK2G inhibition may be a novel therapeutic strategy that overcomes resistance to JAK1/2 inhibition.

Accurately estimating the genetic parameters and revealing more genetic variants underlying milk production and quality are conducive to the genetic improvement of dairy cows. In this study, we estimate the genetic parameters of five milk-related traits of cows-namely, milk yield (MY), milk fat percentage (MFP), milk fat yield (MFY), milk protein percentage (MPP), and milk protein yield (MPY)-based on a random regression test-day model. A total of 95,375 test-day records of 9,834 cows in the lower reaches of the Yangtze River were used for the estimation. In addition, genome-wide association studies (GWASs) for these traits were conducted, based on adjusted phenotypes. The heritability, as well as the standard errors, of MY, MFP, MFY, MPP, and MPY during lactation ranged from 0.22 ± 0.02 to 0.31 ± 0.04, 0.06 ± 0.02 to 0.15 ± 0.03, 0.09 ± 0.02 to 0.28 ± 0.04, 0.07 ± 0.01 to 0.16 ± 0.03, and 0.14 ± 0.02 to 0.27 ± 0.03, respectively, and the genetic correlations between different days in milk (DIM) within lactations decreased as the time interval increased. Two, six, four, six, and three single nucleotide polymorphisms (SNPs) were detected, which explained 5.44, 12.39, 8.89, 10.65, and 7.09% of the phenotypic variation in MY, MFP, MFY, MPP, and MPY, respectively. Ten Kyoto Encyclopedia of Genes and Genomes pathways and 25 Gene Ontology terms were enriched by analyzing the nearest genes and genes within 200 kb of the detected SNPs. Moreover, 17 genes in the enrichment results that may play roles in milk production and quality were selected as candidates, including CAMK2G, WNT3A, WNT9A, PLCB4, SMAD9, PLA2G4A, ARF1, OPLAH, MGST1, CLIP1, DGAT1, PRMT6, VPS28, HSF1, MAF1, TMEM98, and F7. We hope that this study will provide useful information for in-depth understanding of the genetic architecture of milk production and quality traits, as well as contribute to the genomic selection work of dairy cows in the lower reaches of the Yangtze River.

Keywords: Chinese holstein; genetic parameters; genome-wide association study (GWAS); milk-related traits; test-day model.

Aiming at exploring vascular components in multiple sclerosis (MS) with brain outflow disturbance, we combined transcriptome analysis in MS internal jugular vein (IJV) wall with WES in MS families with vertical transmission of disease. Main results were the differential expression in IJV wall of 16 MS-GWAS genes and of seven genes (GRIN2A, GRIN2B, IL20RB, IL26, PER3, PITX2, and PPARGC1A) not previously indicated by GWAS but encoding for proteins functionally interacting with MS candidate gene products. Strikingly, 22/23 genes have been previously associated with vascular or neuronal traits/diseases, nine encoded for transcriptional factors/regulators and six (CAMK2G, GRIN2A, GRIN2B, N1RD1, PER3, PPARGC1A) for circadian entrainment/rhythm components. Among the WES low-frequency (MAF ≤ 0.04) SNPs (n = 7) filtered in the 16 genes, the NR1D1 rs17616365 showed significantly different MAF in the Network for Italian Genomes affected cohort than in the 1000 Genome Project Tuscany samples. This pattern was also detected in five nonintronic variants (GRIN2B rs1805482, PER3 rs2640909, PPARGC1A rs2970847, rs8192678, and rs3755863) in genes coding for functional partners. Overall, the study proposes specific markers and low-frequency variants that might help (i) to understand perturbed biological processes in vascular tissues contributing to MS disease, and (ii) to characterize MS susceptibility genes for functional association with disease-pathways.

Keywords: GWAS; WES; circadian entrainment; circadian rhythm; multiple sclerosis; rare variants; transcriptomics; vascular components.

Platinum resistance accounts for much of the high mortality and morbidity associated with ovarian cancer. Identification of targets with significant clinical translational potential remains an unmet challenge. Through a high-throughput synthetical lethal screening for clinically relevant targets using 290 kinase inhibitors, we identify calcium/calmodulin-dependent protein kinase II gamma (CAMK2G) as a critical vulnerability in cisplatin-resistant ovarian cancer cells. Pharmacologic inhibition of CAMK2G significantly sensitizes ovarian cancer cells to cisplatin treatment in vitro and in vivo. Mechanistically, CAMK2G directly senses ROS, both basal and cisplatin-induced, to control the phosphorylation of ITPKB at serine 174, which directly regulates ITPKB activity to modulate cisplatin-induced ROS stress. Thereby, CAMK2G facilitates the adaptive redox homeostasis upon cisplatin treatment and drives cisplatin resistance. Clinically, upregulation of CAMK2G activity and ITPKB pS174 correlates with cisplatin resistance in human ovarian cancers. This study reveals a key kinase network consisting of CAMK2G and ITPKB for ROS sense and scavenging in ovarian cancer cells to maintain redox homeostasis, offering a potential strategy for cisplatin resistance treatment.