The IGROVCDDP cisplatin-resistant ovarian cancer cell line is an unusual model, as it is also cross-resistant to paclitaxel. IGROVCDDP, therefore, models the resistance phenotype of serous ovarian cancer patients who have failed frontline platinum/taxane chemotherapy. IGROVCDDP has also undergone epithelial-mesenchymal transition (EMT). We aim to determine if alterations in EMT-related genes are related to or independent from the drug-resistance phenotypes. EMT gene and protein markers, invasion, motility and morphology were investigated in IGROVCDDP and its parent drug-sensitive cell line IGROV-1. ZEB1 was investigated by qPCR, Western blotting and siRNA knockdown. ZEB1 was also investigated in publicly available ovarian cancer gene-expression datasets. IGROVCDDP cells have decreased protein levels of epithelial marker E-cadherin (6.18-fold, p = 1.58e-04) and higher levels of mesenchymal markers vimentin (2.47-fold, p = 4.43e-03), N-cadherin (4.35-fold, p = 4.76e-03) and ZEB1 (3.43-fold, p = 0.04). IGROVCDDP have a spindle-like morphology consistent with EMT. Knockdown of ZEB1 in IGROVCDDP does not lead to cisplatin sensitivity but shows a reversal of EMT-gene signalling and an increase in cell circularity. High ZEB1 gene expression (HR = 1.31, n = 2051, p = 1.31e-05) is a marker of poor overall survival in high-grade serous ovarian-cancer patients. In contrast, ZEB1 is not predictive of overall survival in high-grade serous ovarian-cancer patients known to be treated with platinum chemotherapy. The increased expression of ZEB1 in IGROVCDDP appears to be independent of the drug-resistance phenotypes. ZEB1 has the potential to be used as biomarker of overall prognosis in ovarian-cancer patients but not of platinum/taxane chemoresistance.

MicroRNAs (miRNAs) may contribute to the development of depression and its treatment. Here, we used the hypothesis-neutral approach of next-generation sequencing (NGS) to gain comprehensive understanding of the effects of a course of electroconvulsive stimulation (ECS), the animal model equivalent of electroconvulsive therapy (ECT), on rat hippocampal miRNAs. Significant differential expression (p < 0.001) of six hippocampal miRNAs was noted following NGS, after correcting for multiple comparisons. Three of these miRNAs were upregulated (miR-132, miR-212, miR-331) and three downregulated (miR-204, miR-483, miR-301a). qRT-PCR confirmed significant changes in four of the six miRNAs (miR-132, miR-212, miR-204, miR-483). miR-483 was also significantly reduced in frontal cortex, though no other significant alterations were noted in frontal cortex, cerebellum, or whole blood. Assessing the translatability of the results, miR-132 and miR-483 were significantly reduced in whole blood samples from medicated patients with depression (n = 50) compared to healthy controls (n = 45), though ECT had no impact on miRNA levels. Notably, pre-ECT miR-204 levels moderately positively correlated with depression severity at baseline and moderately negatively correlated with mood score reduction post-ECT. miRNAs were also examined in cerebrospinal fluid and serum from a separate cohort of patients (n = 8) treated with ECT; no significant changes were noted post-treatment. However, there was a large positive correlation between changes in miR-212 and mood score post-ECT in serum. Though replication studies using larger sample sizes are required, alterations in miRNA expression may be informative about the mechanism of action of ECS/ECT and in turn might give insight into the neurobiology of depression.

Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.

Compensatory growth (CG) is a naturally occurring phenomenon where, following a period of under nutrition, an animal exhibits accelerated growth upon realimentation. The objective was to identify and quantify hepatic proteins involved in the regulation of CG in cattle. Forty Holstein Friesian bulls were equally assigned to one of four groups. Groups; A1 and A2 had ad libitum access to feed for 125 days, groups R1 and R2 were feed restricted. Following this, R1 and A1 animals were slaughtered. Remaining animals (R2 and A2) were slaughtered following ad libitum feeding for a successive 55 days. At slaughter hepatic tissue samples were collected and label-free quantitative proteomics undertaken with spectra searched against a custom built transcriptome database specific to the animals in this study. 24 differentially abundant proteins were identified during CG (R2 vs. R1) including; PSPH, ASNS and GSTM1, which are involved in nutrient metabolism, immune response and cellular growth. Proteins involved in biochemical pathways related to nutrient metabolism were down-regulated during CG, indicating a possible adaptive response by the liver to a period of fluctuating nutrient availability. The livers ability to regulate its metabolic activity may have profound effects on the efficiency of whole body energy utilization during CG. Significance: This study is the first to unravel the effect of compensatory growth on the hepatic proteome of cattle using transcriptome-assisted shot gun proteomics. Proteins identified as being affected by dietary restriction and subsequent expression of compensatory growth in this study may, following appropriate validation, contribute to the identification of functional genetic variants. Such information could be harnessed within the context of genomic selection in cattle breeding programs to identify animals with a greater genetic potential to undergo compensatory growth, thus increasing the profitability of the beef sector and accelerating genetic gain.

Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alterative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcome and prevent the development of chemoresistance.

Prostate cancer accounts for approximately 13.5% of all newly diagnosed male cancer cases. Significant clinical burdens remain in terms of ineffective prognostication, with overtreatment of insignificant disease. Additionally, the pathobiology underlying disease heterogeneity remains poorly understood. As the role of cancer stem cells in the perpetuation of aggressive carcinoma is being substantiated by experimental evidence, it is crucially important to understand the molecular mechanisms, which regulate key features of cancer stem cells. We investigated two methods for in vitro cultivation of putative prostate cancer stem cells based on 'high-salt agar' and 'monoclonal cultivation'. Data demonstrated 'monoclonal cultivation' as the superior method. We demonstrated that 'holoclones' expressed canonical stem markers, retained the exclusive ability to generate poorly differentiated tumours in NOD/SCID mice and possessed a unique mRNA-miRNA gene signature. miRNA:Target interactions analysis visualised potentially critical regulatory networks, which are dysregulated in prostate cancer holoclones. The characterisation of this tumorigenic population lays the groundwork for this model to be used in the identification of proteomic or small non-coding RNA therapeutic targets for the eradication of this critical cellular population. This is significant, as it provides a potential route to limit development of aggressive disease and thus improve survival rates.

BackgroundFeed efficiency is an important economic and environmental trait in beef production, which can be measured in terms of residual feed intake (RFI). Cattle selected for low-RFI (feed efficient) have similar production levels but decreased feed intake, while also emitting less methane. RFI is difficult and expensive to measure and is not widely adopted in beef production systems. However, development of DNA-based biomarkers for RFI may facilitate its adoption in genomic-assisted breeding programmes. Cattle have been shown to re-rank in terms of RFI across diets and age, while also RFI varies by breed. Therefore, we used RNA-Seq technology to investigate the hepatic transcriptome of RFI-divergent Charolais (CH) and Holstein-Friesian (HF) steers across three dietary phases to identify genes and biological pathways associated with RFI regardless of diet or breed.ResultsResidual feed intake was measured during a high-concentrate phase, a zero-grazed grass phase and a final high-concentrate phase. In total, 322 and 33 differentially expressed genes (DEGs) were identified across all diets for CH and HF steers, respectively. Three genes, GADD45G, HP and MID1IP1, were differentially expressed in CH when both the high-concentrate zero-grazed grass diet were offered. Two canonical pathways were enriched across all diets for CH steers. These canonical pathways were related to immune function.ConclusionsThe absence of common differentially expressed genes across all dietary phases and breeds in this study supports previous reports of the re-ranking of animals in terms of RFI when offered differing diets over their lifetime. However, we have identified biological processes such as the immune response and lipid metabolism as potentially associated with RFI divergence emphasising the previously reported roles of these biological processes with respect to RFI.

Bovine Respiratory Disease (BRD) is the leading cause of mortality in calves. The objective of this study was to examine the response of the host's bronchial lymph node transcriptome to Bovine Respiratory Syncytial Virus (BRSV) in a controlled viral challenge. Holstein-Friesian calves were either inoculated with virus (103.5 TCID50/ml × 15 ml) (n = 12) or mock challenged with phosphate buffered saline (n = 6). Clinical signs were scored daily and blood was collected for haematology counts, until euthanasia at day 7 post-challenge. RNA was extracted and sequenced (75 bp paired-end) from bronchial lymph nodes. Sequence reads were aligned to the UMD3.1 bovine reference genome and differential gene expression analysis was performed using EdgeR. There was a clear separation between BRSV challenged and control calves based on gene expression changes, despite an observed mild clinical manifestation of the disease. Therefore, measuring host gene expression levels may be beneficial for the diagnosis of subclinical BRD. There were 934 differentially expressed genes (DEG) (p < 0.05, FDR <0.1, fold change >2) between the BRSV challenged and control calves. Over-represented gene ontology terms, pathways and molecular functions, among the DEG, were associated with immune responses. The top enriched pathways included interferon signaling, granzyme B signaling and pathogen pattern recognition receptors, which are responsible for the cytotoxic responses necessary to eliminate the virus.

Most forms of castration-resistant prostate cancer (CRPC) are dependent on the androgen receptor (AR) for survival. While, enzalutamide provides a substantial survival benefit, it is not curative and many patients develop resistance to therapy. Although not yet fully understood, resistance can develop through a number of mechanisms, such as AR copy number gain, the generation of splice variants such as AR-V7 and mutations within the ligand binding domain (LBD) of the AR. circular RNAs (circRNAs) are a novel type of non-coding RNA, which can regulate the function of miRNA, and may play a key role in the development of drug resistance. circRNAs are highly resistant to degradation, are detectable in plasma and, therefore may serve a role as clinical biomarkers. In this study, AR-V7 expression was assessed in an isogenic model of enzalutamide resistance. The model consisted of age matched control cells and two sub-line clones displaying varied resistance to enzalutamide. circRNA profiling was performed on the panel using a high throughout microarray assay. Bioinformatic analysis identified a number of differentially expressed circRNAs and predicted five miRNA binding sites for each circRNA. miRNAs were stratified based on known associations with prostate cancer, and targets were validated using qPCR. Overall, circRNAs were more often down regulated in resistant cell lines compared with control (588 vs. 278). Of particular interest was hsa_circ_0004870, which was down-regulated in enzalutamide resistant cells (p ≤ 0.05, vs. sensitive cells), decreased in cells that highly express AR (p ≤ 0.01, vs. AR negative), and decreased in malignant cells (p ≤ 0.01, vs. benign). The associated parental gene was identified as RBM39, a member of the U2AF65 family of proteins. Both genes were down-regulated in resistant cells (p < 0.05, vs. sensitive cells). This is one of the first studies to profile and demonstrate discrete circRNA expression patterns in an enzalutamide resistant cell line model of prostate cancer. Our data suggests that hsa_circ_0004870, through RBM39, may play a critical role in the development of enzalutamide resistance in CRPC.

High rates of calf mortality in the first 12 months of life, results in significant economic losses in Europe and the USA. Bovine respiratory disease (BRD) accounts for the largest proportion of calf mortality. There is a paucity of literature concerning the host response to BRD. In a controlled challenge study in artificially reared dairy calves [155 (S.D. 14) kg], the influence of the host response to bovine respiratory syncytial virus (BRSV) was examined. At AFBI Holstein-Friesian calves were either challenged with BRSV (n=12) or mock challenged with phosphate buffer saline (n=6). Calves were euthanised on day 7 post-challenge. Bronchial lymph nodes were collected and flash-frozen at −80 °C. RNA was extracted and sent to the University of Missouri’s DNA Core Facility for RNA-Seq library preparation and sequencing. Sequenced reads were adapter trimmed, quality assessed using FastQC and aligned to the bovine genome (UMD 3.1) using STAR. Differential gene expression analysis was performed using EdgeR, and pathway and gene ontology analyses were carried out using g:Profiler and Ingenuity Pathway Analysis (IPA). There was a clear separation between BRSV challenged and control calves based on log2 fold gene expression changes, despite an observed mild clinical manifestation of the disease. There were 934 differentially expressed genes (DEG) (P<0.05, FDR<0.1, fold change >2) between the BRSV challenged and control calves. Over-represented pathways and gene ontology terms among the DEG were associated with immune responses and included: GO:0051607 defense response to virus, the KEGG pathway Influenza A and the IPA pathway Interferon Signaling.

The identification of genes implicated in the divergence of residual feed intake (RFI) and their inclusion in genomic assisted breeding programmes may enable rapid, cost-effective selection of low RFI cattle. However, markers for RFI used for genomic selection must be robust across breed and the various diets that may be offered to beef cattle. The objective of this study was to identify genes associated with RFI divergence in hepatic tissue from two cattle breeds over varying dietary stages using differential gene expression analysis and gene co-expression analysis. Using RNA-Seq, hepatic differential gene expression of Charolais (CH) and Holstein-Friesian (HF) steers divergent in RFI was investigated. Animals were offered three contrasting diets, high-concentrate (H1), zero-grazed grass (ZG) and returned to a high-concentrate diet (H2). Liver biopsies were collected at the end of each dietary phase and RNA-Seq libraries were generated for animals most divergent in RFI (n=43 low RFI, n=47 high RFI). On average, 29.1 million reads were obtained per sample, 97% of generated reads had a quality score greater than 30 and 92% of reads mapped uniquely to the reference genome (UMD3.1). At a false discovery rate of 2, 18 differentially expressed genes (DEGs) were identified across diet for CH and 21 DEGs were observed across diet for HF. Agouti signalling protein(ASIP)was differentially expressed across two dietary phases for CH. No gene was differentially expressed in more than one diet for HF. Gene co-expression analysis was carried out using weighted gene co-expression network analysis. Fifteen modules of genes with similar expression patterns across breed and diet were identified. However, none of these modules were significantly correlated with RFI across diet. Our results highlight the difficulty in identifying robust biomarkers for RFI that are applicable across both diet and breed for beef cattle.

Neonatal calves possess a very immature and naïve immune system and are reliant on the intake of maternal colostrum for passive transfer of immunoglobulins. Variation in colostrum management of beef and dairy calves is thought to affect early immune development. Therefore, the objective of this study was to examine changes in gene expression and investigate molecular pathways involved in the immune-competence development of neonatal Holstein dairy calves and naturally suckled beef calves using next generation RNA-sequencing during the first week of life. Jugular whole blood samples were collected from Holstein (H) dairy calves (n = 8) artificially fed 5% B.W. colostrum, and from beef calves which were the progenies of Charolais-Limousin (CL; n = 7) and Limousin-Friesian beef suckler cows (LF; n = 7), for subsequent RNA isolation. In dairy calves, there was a surge in pro-inflammatory cytokine gene expression possibly due to the stress of separation from the dam. LF calves exhibited early signs of humoral immune development with observed increases in the expression genes coding for Ig receptors, which was not evident in the other breeds by 7 days of age. Immune and health related DEGs identified as upregulated in beef calves are prospective contender genes for the classification of biomarkers for immune-competence development, and will contribute towards a greater understanding of the development of an immune response in neonatal calves.

The objective of this study was to identify transcriptional variants in key genes involved in compensatory growth (CG) in cattle. Fifteen Holstein-Friesian bulls were offered a restricted diet (growing at 0.6kg/day) for 125 days (Period 1) followed by ad libitum access to feed for a further 55 days (Period 2), during which they displayed CG. A control group received ad libitum access to feed across both periods. Following 55 days of ad libitum feeding and CG, tissue samples including liver, rumen papillae and jejunal epithelium were sampled from all animals. RNAseq was performed on tissue samples and transcriptional variants were subsequently called from resultant sequencing data as per the GATK guidelines. The Ensembl Variant Effect Predictor program was then used for annotation of identified variants. The bulls displayed CG during Period 2 growing at 1.8 times that rate of the control group. Of the key genes examined (n=53), 1854 transcript variants were identified in cattle undergoing CG. Of the variants identified, 69% were synonymous, 29% were missense, 1% were frameshift variants while 1% were inframe deletions. Four genes contained high impact variants in the liver, namely: CYP51A1, SEMA4B, ACSM1 and ELN. CYP51A1 and SEMA4B which are involved in cholesterol synthesis and receptor activity respectively contained frameshift variants. Two splicing variants were also present in liver; ACSM1, which is involved in GTP binding and ELN which encodes a major structural protein. These same high impact variants were also evident during CG within the rumen; CYP51A1, SEMA4B and ELN and in the jejunum ELN was also classified as a high impact variant. Transcriptional variants identified in the current study may, following validation, may hold potential for use as DNA based biomarkers for the selection of cattle with a superior ability to undergo CG following dietary restriction.

The rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.

It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10 − /ALDH − CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10 − /ALDH − CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10 − /ALDH − CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10 − /ALDH − CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.

We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.

OBJECTIVE:MicroRNAs are short, non-coding molecules that regulate gene expression. Here, we investigate the role of microRNAs in depression and electroconvulsive therapy (ECT).METHODS:We performed three studies: a deep sequencing discovery-phase study of miRNA changes in whole blood following ECT (n = 16), followed by a validation study in a separate cohort of patients pre-/post-ECT (n = 37) and matched healthy controls (n = 34). Changes in an experimentally validated gene target (VEGFA) were then analysed in patients pre-/post-ECT (n = 97) and in matched healthy controls (n = 53).RESULTS:In the discovery-phase study, we found no statistically significant differences in miRNA expression from baseline to end of treatment in the group as a whole, but post hoc analysis indicated a difference in patients with psychotic depression (n = 3). In a follow-up validation study, patients with psychotic depression (n = 7) had elevated baseline levels of miR-126-3p (t = 3.015, P = 0.006) and miR-106a-5p (t = 2.598, P = 0.025) compared to healthy controls. Following ECT, these differences disappeared. Baseline VEGFA levels were significantly higher in depressed patients compared to healthy controls (F(1,144) = 27.688, P = <0.001). Following ECT, there was a significant change in VEGFA levels in the psychotic group only (t = 2.915, P = 0.010).CONCLUSION:Molecular differences (miRNA and VEGFA) may exist between psychotic and non-psychotic depression treated with ECT.