BACKGROUND

Wilms tumor (WT) is an embryonic kidney cancer, for which histone acetylation might be a therapeutic target. LBH589, a novel targeted agent, suppresses histone deacetylases in many tumors. This study investigated the antitumor activity of LBH589 in SK-NEP-1 and G401 cells.

METHODS

SK-NEP-1 and G401 cell growth was assessed by CCK-8 and in nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometry detected apoptosis in cell culture. Gene expressions of LBH589-treated tumor cells were analyzed using an Arraystar Human LncRNA Array. The Multi Experiment View cluster software analyzed the expression data. Differentially expressed genes from the cluster analyses were imported into the Ingenuity Pathway Analysis tool.

RESULTS

LBH589 inhibited cell proliferation of SK-NEP-1 and G401 cells in a dose-dependent manner. Annexin V, TUNEL and Hochest 33342 staining analysis showed that LBH589-treated cells showed more apoptotic features compared with the control. LBH589 treatment inhibited the growth of SK-NEP-1 xenograft tumors in nude mice. Arraystar Human LncRNA Array analysis of genes and lncRNAs regulated by LBH589 identified 6653 mRNAs and 8135 lncRNAs in LBH589-treated SK-NEP-1 cells. The most enriched gene ontology terms were those involved in nucleosome assembly. KEGG pathway analysis identified cell cycle proteins, including CCNA2, CCNB2, CCND1, CCND2, CDK4, CDKN1B and HDAC2, etc. Ingenuity Pathway Analysis identified important upstream molecules: HIST2H3C, HIST1H4A, HIST1A, HIST1C, HIST1D, histone H1, histone H3, RPRM, HSP70 and MYC.

CONCLUSIONS

LBH589 treatment caused apoptosis and inhibition of cell proliferation of SK-NEP-1and G401 cells. LBH589 had a significant effect and few side effects on SK-NEP-1 xenograft tumors. Expression profiling, and GO, KEGG and IPA analyses identified new targets and a new "network" of genes responding to LBH589 treatment in SK-NEP-1 cells. RPRM, HSP70 and MYC may be important regulators during LBH589 treatment. Our results provide new clues to the proapoptotic mechanism of LBH589.

Recurrent pregnancy loss (RPL) concerns ~3% of couples aiming at childbirth. In the current study, transcriptomes and miRNomes of 1st trimester placental chorionic villi were analysed for 2 RPL cases (≥6 miscarriages) and normal, but electively terminated pregnancies (ETP; n = 8). Sequencing was performed on Illumina HiSeq 2000 platform. Differential expression analyses detected 51 (27%) transcripts with increased and 138 (73%) with decreased expression in RPL compared to ETP (DESeq: FDR P < 0.1 and DESeq2: <0.05). RPL samples had substantially decreased transcript levels of histones, regulatory RNAs and genes involved in telomere, spliceosome, ribosomal, mitochondrial and intra-cellular signalling functions. Downregulated expression of HIST1H1B and HIST1H4A (Wilcoxon test, fc≤0.372, P≤9.37 × 10-4) was validated in an extended sample by quantitative PCR (RPL, n = 14; ETP, n = 24). Several upregulated genes are linked to placental function and pregnancy complications: ATF4, C3, PHLDA2, GPX4, ICAM1, SLC16A2. Analysis of the miRNA-Seq dataset identified no large disturbances in RPL samples. Notably, nearly 2/3 of differentially expressed genes have binding sites for E2F transcription factors, coordinating mammalian endocycle and placental development. For a conceptus destined to miscarriage, the E2F TF-family represents a potential key coordinator in reprogramming the placental genome towards gradually stopping the maintenance of basic nuclear and cellular functions.

Lung cancer remains to be the leading cause of cancer-related mortality worldwide. Finding new noninvasive biomarkers for lung cancer is still a significant clinical challenge. Exosomes are membrane-bound, nano-sized vesicles that are released by various living cells. Studies on exosomal proteomics may provide clues for developing clinical assays. In this study, we performed semi-quantitative proteomic analysis of proteins that were purified from exosomes of NCI-H838 non-small cell lung cancer cell line, with total cellular membrane proteins as control. In the exosomes, LC-MS/MS by data-independent analysis mode identified 3235 proteins. THBS1, ANXA6, HIST1H4A, COL18A1, MDK, SRGN, ENO1, TUBA4A, SLC3A2, GPI, MIF, MUC1, TALDO1, SLC7A5, ICAM1, HSP90AA1, G6PD, and LRP1 were found to be expressed in exosomes at more than 5-fold higher level as compared to total cellular membrane proteins. A well-known cancer biomarker, MUC1, is expressed at 8.98-fold higher in exosomes than total cellular membrane proteins. Subsequent analysis of plasma exosomes from non-small cell lung cancer (NSCLC) patients by a commercial electrochemiluminescence immunoassay showed that exosomal MUC1 level is 1.5-fold higher than healthy individuals (mean value 1.55 ± 0.16 versus mean value 1.05 ± 0.06, p = 0.0213). In contrast, no significant difference of MUC1 level was found between NSCLC patients and healthy individuals' plasma (mean value 5.48 ± 0.65 versus mean value 4.16 ± 0.49). These results suggest that certain proteins, such as MUC1, are selectively enriched in the exosome compartment. The mechanisms for their preferential localization and their biological roles remain to be studied.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and the proteins and pathways involved in the pathophysiology are not fully understood. Even less is known about the preclinical disease phase. To uncover new ALS-related proteins and pathways, we performed a comparative proteomic analysis in cerebrospinal fluid (CSF) of asymptomatic (n = 14) and symptomatic (n = 14) ALS mutation carriers and sporadic ALS patients (n = 12) as well as post-mortem human spinal cord tissue (controls: n = 7, ALS, n = 8). Using a CSF-optimized proteomic workflow, we identified novel (e.g., UCHL1, MAP2, CAPG, GPNMB, HIST1H4A, HIST1H2B) and well-described (e.g., NEFL, NEFH, NEFM, CHIT1, CHI3L1) protein level changes in CSF of sporadic and genetic ALS patients with enrichment of proteins related to transcription, cell cycle and lipoprotein remodeling (total protein IDs: 2303). No significant alteration was observed in asymptomatic ALS mutation carriers representing the prodromal disease phase. We confirmed UCHL1, MAP2, CAPG and GPNMB as novel biomarker candidates for ALS in an independent validation cohort of patients (n = 117) using multiple reaction monitoring. In spinal cord tissue, 292 out of 6810 identified proteins were significantly changed in ALS with enrichment of proteins involved in mRNA splicing and of the neurofilament compartment. In conclusion, our proteomic data in asymptomatic ALS mutation carriers support the hypothesis of a sudden disease onset instead of a long preclinical phase. Both CSF and tissue proteomic data indicate transcriptional pathways to be amongst the most affected. UCHL1, MAP2 and GPNMB are promising ALS biomarker candidates which might provide additional value to the established neurofilaments in patient follow-up and clinical trials.

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8⁺ T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.

Diagnosis of SLE relies on the detection of autoantibodies. We aimed to assess the diagnostic potential of histone H4 and H2A variant antibodies in SLE.

IgG-autoantibodies to histones H4 (HIST1H4A), H2A type 2-A (HIST2H2AA3) and H2A type 2-C (HIST2H2AC) were measured along with a standard antibody (SA) set including SSA, SSB, Sm, U1-RNP and RPLP2 in a multiplex magnetic microsphere-based assay in 153 SLE patients [85% female, 41 (13.5) years] and 81 healthy controls [77% female, 43.3 (12.4) years]. Receiver operating characteristic analysis was performed to assess diagnostic performance of individual markers. Logistic regression analysis was performed on a random split of samples to determine the additional value of histone antibodies in comparison with SA by likelihood ratio test and determination of diagnostic accuracy in the remaining validation samples.

Microsphere-based assay showed good interclass correlation (mean 0.85, range 0.73-0.99) and diagnostic performance in receiver operating characteristic analysis (area under the curve (AUC) range 84.8-93.2) compared with routine assay for SA parameters. HIST1H4A-IgG was the marker with the best individual diagnostic performance for SLE vs healthy (AUC 0.97, sensitivity 95% at 90% specificity). HIST1H4A-IgG was an independent significant predictor for the diagnosis of SLE in multivariate modelling (P < 0.0001), and significantly improved prediction of SLE over SA parameters alone (residual deviance 45.9 vs 97.1, P = 4.3 × 10-11). Diagnostic accuracy in the training and validation samples was 89 and 86% for SA, and 95 and 89% with the addition of HIST1H4A-IgG.

HIST1H4A-IgG antibodies improve diagnostic accuracy for SLE vs healthy.

Cardiomyopathy (CMP) is a group of myocardial diseases that progressively impair cardiac function. The mechanisms underlying CMP development are poorly understood, but lifestyle factors are clearly implicated as risk factors. This study aimed to identify molecular biomarkers involved in inflammatory CMP development and progression using a systems biology approach. The authors analysed microarray gene expression datasets from CMP and tissues affected by risk factors including smoking, ageing factors, high body fat, clinical depression status, insulin resistance, high dietary red meat intake, chronic alcohol consumption, obesity, high-calorie diet and high-fat diet. The authors identified differentially expressed genes (DEGs) from each dataset and compared those from CMP and risk factor datasets to identify common DEGs. Gene set enrichment analyses identified metabolic and signalling pathways, including MAPK, RAS signalling and cardiomyopathy pathways. Protein-protein interaction (PPI) network analysis identified protein subnetworks and ten hub proteins (CDK2, ATM, CDT1, NCOR2, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E and HIST1H4L). Five transcription factors (FOXC1, GATA2, FOXL1, YY1, CREB1) and five miRNAs were also identified in CMP. Thus the authors' approach reveals candidate biomarkers that may enhance understanding of mechanisms underlying CMP and their link to risk factors. Such biomarkers may also be useful to develop new therapeutics for CMP.

BACKGROUND

Although improved understanding and assessment of organ rejection significantly contribute to long-term allograft survival after kidney transplantation, reliable and predictive biomarkers that enable diagnoses of rejection state are lacking. Patient rejection of a kidney graft displays a specific blood and biopsy transcriptional pattern, raising the question of whether transcript biomarkers in blood could reflect events within the allograft.

METHODS

Differential expression genes were screened on large-scale transcriptomic data from blood and allograft biopsies, which included recipients undergoing rejection and recipients with stable renal function.

RESULTS

We found that the number of rejection-related genes in biopsy samples was much greater than in blood. We observed only one overlapping gene, HIST1H4A, consistently expressed in biopsy samples and blood. Functional association of the identified genes in biopsies implicated a strong involvement of inflammatory-immune pathways. Rejection-related genes in the mammalian target of rapamycin-signaling pathway were down-regulated, and genes related to allograft rejection and graft-versus-host disease were up-regulated in allograft biopsy samples. We also recognized the core signaling elements (PIK3R2 and EGFR) in inflammatory-immune pathways based on biopsy samples.

CONCLUSIONS

We have expanded our understanding of rejection-specific gene expression pattern in allograft biopsy and peripheral blood, and provided a candidate set of overlapping genes for screening of rejection in kidney transplant recipients.

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high-throughput approach to identify these isoforms. Using an oversimplified top-down LC-MS/MS strategy, we detected, around the 26-kD position of SDS-PAGE, proteins produced from 782 genes in a Cdk4-/- mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one-fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, i.e. between 23-29 kD, the range set as allowed variation in SDS-PAGE. These 213 proteins are considered as the wild type (WT). The remaining three-fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two-thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger-group or a smaller-group, respectively, for their appearance at a higher or lower position of SDS-PAGE. For instance, at this 26-kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4 and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS-PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4-/- cell line, thus wondering whether some of other gene-knockout cells or organisms show similar incompleteness of the knockout. This article is protected by copyright. All rights reserved.

Spinal cord injury (SCI) is a common but severe disease caused by traffic accidents. Coronary atherosclerotic heart disease (CHD) caused by dyslipidemia is known as the leading cause of death in patients with SCI. However, the quantitative analysis showed that the cholesterol and lipoprotein concentrations in peripheral blood (PB) did not change significantly within 48 h after SCI. Due to the presence of the Blood spinal cord barrier (BSCB), there are only few studies concerning the plasma cholesterol metabolism in the acute phase of SCI. Exosomes have a smaller particle size, which enables them relatively less limitation of BSCB. This study uses exosomes derived from the plasma of 43 patients in the acute phase of SCI and 71 patients in the control group as samples. MS proteomics and bioinformatics analysis found 590 quantifiable proteins, in which 75 proteins were upregulated and 153 proteins were downregulated, and the top 10 differentially expressed proteins are those including downregulating proteins: HIST1H4A, HIST2H3A, HIST2H2BE, HCLS1, S100A9, HIST1H2BM, S100A8, CALM3, YWHAH, and SFN, and upregulating proteins: SERPIND1, C1QB, SPTLC3, IGHV4-28, C4A, IGHV4-38-2, IGHV4-30-2, SLC15A1, C4B, and ACTG2. Enrichment analysis showed that the largest part of proteins was related to cholesterol metabolism among the downregulated proteins. The main components of cholesterol [ApoB-48 and ApoB-100 increased, ApoA-I, ApoA-II, ApoA-IV, ApoC, ApoE, and Apo(a) decreased] were changed in exosomes derived from plasma of patients. ELISA analysis showed that some components were disordered in the acute phase of SCI. These results suggested that the exosomes might be involved in cholesterol metabolism regulation in the acute phase of SCI.

Keywords: bioinformatic analysis; cholesterol metabolism; exosome; proteomics; spinal cord injury.

The Theranostics approach has full potential to completely transform the contemporary medicine system to a patient-centric approach, as it is emerging in quite efficient manner, over the past few years. The primary impetus of this review is to analyse the patent growth in the domain of breast cancer theranostics. This wholesome analysis provides an insight into the current technological and R & D advancement over the years, in breast cancer theranostics. Thus, guide the end-users in getting the conclusion for policymaking and other public recommendations. This patent assessment also foretells about the future trends to carry out further achievements. Due to their easy availability, information richness, & versatility, patent's role in R&D policy has been emphasized by stake holders of innovation including scientists time to time. Graphical Abstract: The figure illustrates the applied technologies used for breast cancer theranostics by top three forward cited patents (A) The oligonucleotides with specific sequences (comprised of at least one of DNA, RNA, PNA. LNA, UNA or combination)¹ are capable of binding a targeted tumor protein (PARP1, HISTIHIB, HISTIHID, NCL, FBL, SFPQ, RPL12, ACTB, HIST1H4A, SSBP1, NONO, H2AFJ, and DDX21, forming a tumor protein complex or subunit or their fragments and might block the tumoral activity. These are also capable of binding to Ramos cells (Derived from Human Burkitt's lymphoma that is negative for Epstein Barr virus). These can also bind cell surface nucleolin and may inhibit cell proliferation. These molecules with detection agent detect the presence or level of disease specific protein. (B) These aptamers with chemical functionalization can be conjugated to an amine linker or high molecular weight non-immunogenic compound or a drug or cytotoxic moiety or labelled with fluorescent agent. These chemically modified aptamers can also bind disease - specific biomarkers e.g., circulating biomarkers, micro- vesicle surface antigens or their functional fragments and can be subsequently used for early diagnosis, prognosis or therapeutic purposes. ¹PNA: Peptide Nucleic Acid. LNA: Locked Nucleic Acid. UNA: Unlocked Nucleic Acid.

Keywords: Diagnostics; Nanomedicine; Nanotechnology; Patent; Theranostics.