BACKGROUND

With the development of sequencing technologies, there may be some disputes on sequencing analysis. The aim of this study was to investigate different allele frequency thresholds of mutations in targeted genes on prognostic analyses using a panel of cancer associated gene exons (CAGE) in oral squamous cell carcinoma (OSCC).

METHODS

Forty-six patients were included in this study. Twelve genes were sequenced and analyzed using next-generation sequencing from formalin-fixed paraffin-embedded tissues. Allele frequency thresholds of 10, 5, and 3% were used for prognostic analyses.

RESULTS

With a mean sequence depth of 3199-fold, 99% of CAGE were represented by at least 10 reads. Ninety-four non-synonymous (missense [70.2%], nonsense [11.7%], splice site [10.6%], and insertion/deletion [7.5%]) mutations were detected in 40 OSCC patients with an allele frequency threshold of 10%. TP53 (78.3%), NOTCH1 (30.4%), CASP8 (13.0%), CDKN2A (10.9%), and CDH1 (6.5%) were the most frequently mutated genes. Using allele frequency thresholds of 10, 5, and 3%, there were no significant differences in clinical outcomes between patients with non-synonymous mutations and wild type genotypes.

CONCLUSIONS

TP53, NOTCH1, CASP8, CDKN2A, and CDH1 are the most frequently mutated genes in OSCC patients. The allele frequency threshold used in this study does not affect the results of clinical outcome analysis.

It is hypothesized that same driver gene mutations should be present in both oral leukoplakia and cancer tissues. So, we attempted to find out mutations at one of the driver genes, CASP8, in cancer and adjacent leukoplakia tissues. Patients (n = 27), affected by both of cancer and adjacent leukoplakia, were recruited for the study. Blood and tissue DNA samples were used to identify somatic mutations at CASP8 by next generation sequencing method. In total, 56% (15 out of 27) cancer and 30% (8 out of 27) leukoplakia tissues had CASP8 somatic mutations. In 8 patients, both cancer and adjacent leukoplakia tissues, located within 2-5 cm of tumor sites, had identical somatic mutations. But, in 7 patients, cancer samples had somatic mutations but none of the leukoplakia tissues, located beyond 5cm of tumor sites, had somatic mutations. Mutated allele frequencies at CASP8 were found to be more in cancer compared to adjacent leukoplakia tissues. This study provides mutational evidence that oral cancer might have progressed from previously grown leukoplakia lesion. Leukoplakia tissues, located beyond 5cm of cancer sites, were free from mutation. The study implies that CASP8 mutation could be one of the signatures for some of the leukoplakia to progress to oral cancer.

The antioxidant and neuroprotective activity of Glucomoringin isothiocyanate (GMG-ITC) have been reported in in vivo and in vitro models of neurodegenerative diseases. However, its neuroprotective role via mitochondrial-dependent pathway in a noxious environment remains unknown. The main objective of the present study was to unveil the mitochondrial apoptotic genes' profile and prospectively link with neuroprotective activity of GMG-ITC through its ROS scavenging. The results showed that pre-treatment of differentiated SH-SY5Y cells with 1.25 μg/mL purified isolated GMG-ITC, significantly reduced reactive oxygen species (ROS) production level, compared to H₂O₂ control group, as evidenced by flow cytometry-based evaluation of ROS generation. Presence of GMG-ITC prior to development of oxidative stress condition, downregulated the expression of cyt-c, p53, Apaf-1, Bax, CASP3, CASP8 and CASP9 genes with concurrent upregulation of Bcl-2 gene in mitochondrial apoptotic signalling pathway. Protein Multiplex revealed significant decreased in cyt-c, p53, Apaf-1, Bax, CASP8 and CASP9 due to GMG-ITC pre-treatment in oxidative stress condition. The present findings speculated that pre-treatment with GMG-ITC may alleviate oxidative stress condition in neuronal cells by reducing ROS production level and protect the cells against apoptosis via neurodegenerative disease potential pathways.

Anorexia nervosa (AN) is a severe psychiatric disorder with high mortality and relapse rates. Even though changes in inflammatory markers and cytokines are known to accompany cachexia associated with somatic disorders such as cancer and chronic kidney disorder, studies on inflammatory markers in AN are rare and typically include few individuals. Here, we utilize an Olink Proteomics inflammatory panel to explore the concentrations of 92 preselected inflammation-related proteins in plasma samples from women with active AN (N=113), recovered from AN (AN-REC, N=113), and normal weight healthy controls (N=114). After correction for multiple testing, twenty-five proteins differed significantly between the AN group and controls (lower levels: ADA, CCL19, CD40, CD5, CD8A, CSF1, CXCL1, CXCL5, HGF, IL10RB, IL12B, 1L18R1, LAP TGFß1, MCP3, OSM, TGFα, TNFRSF9, TNFS14 and TRANCE; higher levels: CCL11, CCL25, CST5, DNER, LIFR and OPG). Although more than half of these differences (N=15) were present in the comparison between AN and AN-REC, no significant differences were seen between AN-REC and controls. Furthermore, twenty-five proteins correlated positively with BMI (ADA, AXIN1, CASP8, CD5, CD40, CSF1, CXCL1, CXCL5, EN-RAGE, HGF, IL6, IL10RB, IL12B, IL18, IL18R1, LAP TGFß1, OSM, SIRT2, STAMBP, TGFα, TNFRSF9, TNFS14, TRANCE, TRAIL and VEGFA) and four proteins correlated negatively with BMI (CCL11, CCL25, CCL28 and DNER). These results suggest that a dysregulated inflammatory status is associated with AN, but, importantly, seem to be confined to the acute illness state.

The aim of the present study was to reveal the potential molecular mechanisms of microsatellite instability (MSI) on the prognosis of gastric cancer (GC). The investigation was performed based on an RNAseq expression profiling dataset downloaded from The Cancer Genome Atlas, including 64 high‑level MSI (MSI‑H) GC samples, 44 low‑level MSI (MSI‑L) GC samples and 187 stable microsatellite (MSI‑S) GC samples. Differentially expressed genes (DEGs) were identified between the MSI‑H, MSI‑L and MSI‑S samples. Pathway enrichment analysis was performed for the identified DEGs and the pathway deviation scores of the significant enrichment pathways were calculated. A Multi‑Layer Perceptron (MLP) classifier, based on the different pathways associated with the MSI statuses was constructed for predicting the outcome of patients with GC, which was validated in another independent dataset. A total of 190 DEGs were selected between the MSI‑H, MSI‑L and MSI‑S samples. The MLP classifier was established based on the deviation scores of 10 significant pathways, among which antigen processing and presentation, and inflammatory bowel disease pathways were significantly enriched with HLA‑DRB5, HLA‑DMA, HLA‑DQA1 and HLA‑DRA; the measles, toxoplasmosis and herpes simplex infection pathways were significantly enriched with Janus kinase 2 (JAK2), caspase‑8 (CASP8) and Fas. The classifier performed well on an independent validation set with 100 GC samples. Taken together, the results indicated that MSI status may affect GC prognosis, partly through the antigen processing and presentation, inflammatory bowel disease, measles, toxoplasmosis and herpes simplex infection pathways. HLA‑DRB5, HLA‑DMA, HLA‑DQA1, HLA‑DRA, JAK2, CASP8 and Fas may be predictive factors for prognosis in GC.

Apoptosis plays an important role in controlling the adaptive immune response and general homeostasis of the immune cells, and impaired apoptosis in the immune system results in autoimmunity and immune dysregulation. In the last 25 years, inherited human diseases of the Fas-FasL pathway have been recognized. Autoimmune lymphoproliferative syndrome (ALPS) is an inborn error of immunity, characterized clinically by nonmalignant and noninfectious lymphoproliferation, autoimmunity, and increased risk of lymphoma due to a defect in lymphocyte apoptosis. The laboratory hallmarks of ALPS are an elevated percentage of T-cell receptor αβ double negative T cells (DNTs), elevated levels of vitamin B12, soluble FasL, IL-10, IL-18 and IgG, and defective in vitro Fas-mediated apoptosis. In order of frequency, the genetic defects associated with ALPS are germinal and somatic ALPS-FAS, ALPS-FASLG, ALPS-CASP10, ALPS-FADD, and ALPS-CASP8. Partial disease penetrance and severity suggest the combination of germline and somatic FAS mutations as well as other risk factor genes. In this report, we summarize human defects of apoptosis leading to ALPS and defects that are known as ALPS-like syndromes that can be clinically similar to, but are genetically distinct from, ALPS. An efficient genetic and immunological diagnostic approach to patients suspected of having ALPS or ALPS-like syndromes is essential because this enables the establishment of specific therapeutic strategies for improving the prognosis and quality of life of patients.

Keywords: ALPS; ALPS-like; apoptosis; autoimmunity; immune dysregulation.

BACKGROUND

Lysophosphatidic acid (LPA) regulates reproductive processes in the cow. Ovarian granulosa cells play a pivotal role in follicle growth and development. Nevertheless, the role of LPA in the local regulation of granulosa cell function in different follicle categories in the bovine ovary has not been investigated.

METHODS

Ovarian follicles were divided into healthy, transitional and atretic categories. The expression levels of AX, PLA2, LPARs and factors involved in apoptosis and cell survival processes in granulosa cells in different types of follicles were measured by real-time PCR. The correlations between the expression levels of AX, PLA2, LPARs and the examined factors were measured. The immunolocalization of AX, PLA2 and LPARs in different ovarian follicles was examined by immunohistochemistry. Statistical analyses were conducted in GraphPad using a one-way ANOVA followed by the Kruskal-Wallis multiple comparison test or a correlation analysis followed by Pearson's test.

RESULTS

The expression levels of AX, PLA2 and LPARs, with the major role of LPAR2 and PLA2, were found in the granulosa cells originating from different follicle types. The expression levels of the factors involved in cell apoptosis (TNFα and its receptors, FAS, FASL, CASP3, CASP8, β-glycan, and DRAK2) were significantly higher in the granulosa cells of the atretic follicles compared to the healthy follicles. A number of correlations between LPARs, AX, PLA2 and factors associated with apoptosis were observed in the atretic but not in the healthy follicles. A greater expression of the factors involved in differentiation and proliferation in the granulosa cells (DICE1 and SOX2) was found in the healthy follicles in comparison with the atretic. A number of correlations between LPARs, AX, PLA2 and the factors associated with cell survival were observed in the healthy but not in the atretic follicles.

CONCLUSIONS

Granulosa cells are the target of LPA action and the source of LPA synthesis in the bovine ovarian follicle. We suggest that the participation of LPA in apoptosis in the atretic follicles mainly occurs through the regulation of TNF-α-dependent and caspase-induced pathways. In the transitional follicles, LPA might influence the inhibins to shift the balance between the number of healthy and atretic follicles. In the healthy follicle type, LPA, acting via LPAR1, might regulate MCL1 and estradiol-stimulating ERβ mRNA expression, leading to the stimulation of anti-apoptotic processes in the granulosa cells and their differentiation and proliferation.

A novel clustering approach identified five subgroups of diabetes with distinct progression trajectories of complications. We hypothesized that these subgroups differ in multiple biomarkers of inflammation. Serum levels of 74 biomarkers of inflammation were measured in 414 individuals with recent adult-onset diabetes from the German Diabetes Study (GDS) allocated to five subgroups based on data-driven analysis. Pairwise differences between subgroups for biomarkers were assessed with generalized linear mixed models before (model 1) and after adjustment (model 2) for the clustering variables. Participants were assigned to five subgroups: severe autoimmune diabetes (SAID, 21%), severe insulin-deficient diabetes (SIDD, 3%), severe insulin-resistant diabetes (SIRD, 9%), mild obesity-related diabetes (MOD, 32%) and mild age-related diabetes (MARD, 35%). In model 1, 23 biomarkers showed ≥1 pairwise difference between subgroups (Bonferroni-corrected p<0.0007). Biomarker levels were generally highest in SIRD and lowest in SIDD. All 23 biomarkers correlated with ≥1 of the clustering variables. In model 2, three biomarkers (CASP-8, EN-RAGE, IL-6) showed at least one pairwise difference between subgroups (e.g. lower CASP8, EN-RAGE and IL-6 in SIDD vs. all other subgroups, all p<0.0007). Thus, novel diabetes subgroups show multiple differences in biomarkers of inflammation, underlining a prominent role of inflammatory pathways in particular in SIRD.

KLK6 is a serine protease normally expressed in mammary tissue, and aberrantly regulated in breast cancer. At physiological levels, KLK6 functions as a suppressor of breast cancer, while its aberrant overexpression (>50-fold higher than normal) is characteristic of a subset of breast cancers and has been linked to accelerated growth of primary breast tumors in SCID mice (Pampalakis et al. Cancer Res 2009). Here, we investigated the molecular mechanisms underlying the concentration-dependent functions of KLK6 by comparing MDA-MB-231 stable transfectants expressing increasing levels of KLK6 in in vitro and in vivo tumorigenicity assays (soft agar, xenograft growth, tail vein metastasis). Quantitative proteomics was applied to identify proteins that are altered upon re-expression of KLK6 in MDA-MB-231 at normal or constitutive levels. Overexpression of KLK6 is associated with increased metastatic ability of breast cancer cells into lungs, increased expression of certain S100 proteins (S100A4, S100A11) and keratins, and downregulation of the apoptosis-related proteases CASP7 and CASP8, and RABs. On the other hand, KLK6 re-expression at physiological levels leads to inhibition of lung metastases associated with suppression of S100 proteins (S100A4, S100A10, S100A13, S100A16) and induced CASP7 and CASP8 expression. As this is the first report that KLK6 expression is associated with S100 proteins, caspases, RABs and keratins, we validated this finding in clinical datasets. By integrating proteomics and microarray data from breast cancer patients, we generated two composite scores, (KLK6+S100B-S100A7) and (KLK6+S100B-S100A14-S100A16), to predict long-term survival of breast cancer patients. We present previously unknown pathways implicating KLK6 in breast cancer. The findings promise to aid our understanding of the functional roles of KLK6 in breast cancer and may yield new biomarkers for the cancer types in which KLK6 is known to be aberrantly up-regulated.

A human neuroblastoma cell line, NB-1, was treated with 24 h of microgravity simulation by clinostat, or irradiated with extremely small X-ray doses of 0.1 or 1.0 mGy using single and 10 times fractionation regimes with 1 and 2 h time-intervals. A quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) examination was performed for apoptosis related factors (BAX, CYTC, APAF1, VDAC1-3, CASP3, CASP8, CASP9 P53, AIF, ANT1 and 2, BCL2, MnSOD, autophagy related BECN and necrosis related CYP-40. The qRT-PCR results revealed that microgravity did not result in significant changes except for a upregulation of proapoptotic VDAC2, and downregulations of proapoptotic CASP9 and antiapoptotic MnSOD. After 0.1 mGy fractionation irradiation, there was increased expression of proapoptotic APAF1 and downregulation of proapoptotic CYTC, VDAC2, VDAC3, CASP8, AIF, ANT1, and ANT2, as well as an increase in expression of antiapoptotic BCL2. There was also a decrease in MnSOD expression with 0.1 mGy fractionation irradiation. These results suggest that microgravity and low-dose radiation may decrease apoptosis but may potentially increase oxidative stress.

Background: Multidrug resistance (MDR) causes failure of doxorubicin therapy of cancer cells, which develops after or during doxorubicin treatment resulting in cross-resistance to structurally and functionally-unrelated other anticancer drugs. MDR is multifactorial phenomenon associated with overexpression of ATP-binding cassette (ABC) transporters, metabolic enzymes, impairment of apoptosis, and alteration of cell cycle checkpoints. The cancer-prevention of the dietary carotenoid; fucoxanthin (FUC) has been extensively explored. Nevertheless, the underlying mechanism of its action is not full elucidated.

Hypothesis/purpose: Investigation of the underlying mechanism of MDR reversal by the dietary carotenoid fucoxanthin (FUC) and its ability to enhance the doxorubicin (DOX) cytotoxicity in resistant breast (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) cell lines.

Methods: The synergistic interaction of FUC and DOX was evaluated using several techniques, viz.; MTT assay, ABC transporter function assays using FACS and fluorimetry, enzyme activity via spectroscopy and luminescence assays, and apoptosis assay using FACS, and gene expression using RTPCR.

Results: FUC (20 µM) synergistically enhanced the cytotoxicity of DOX and significantly reduced the dose of DOX (FR) in DOX resistant cells (MCF-7/ADR), hepatic (HepG-2/ADR), and ovarian (SKOV-3/ADR) to 8.42-(CI= 0.25), 6.28-(CI= 0.32), and 4.56-fold (CI=0.37) (P<0.001). FUC significantly increased the accumulation of DOX more than verapamil in resistant cells by 2.70, 2.67, and 3.95-fold of untreated cells (p<0.001), respectively. A FUC and DOX combination significantly increased the Rho123 accumulation higher than individual drugs by 2.36-, 2.38-, 1.89-fold verapamil effects in tested cells (p<0.001), respectively. The combination of the FUC and DOX decreased ABCC1, ABCG2, and ABCB1 expression. The FUC and DOX combination increased the levels and activity of caspases (CASP3, CASP8) and p53, while decreased the levels and activity of CYP3A4, GST, and PXR in resistant cancer cells. The combination induced early/late apoptosis to 91.9/5.4% compared with 0.0/0.7% of untreated control.

Conclusion: Our data suggests a new dietary and therapeutic approach of combining the FUC with DOX to overcome multidrug resistance in cancer cells. However, animal experiments should be conducted to confirm the findings before applying the results into clinical trials.

Keywords: Carotenoids; Chemoprevention of cancer; DOX; Fucoxanthin; Multidrug resistance.

Aims: Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signaling and a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodeling in experimental RHD.

Methods and results: Pulmonary hypertension (PH) and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting one day before MCT administration. Right-atrial (RA) conduction and gene-expression were analyzed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary-artery or right-ventricular remodeling. Nevertheless, in vivo transesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P < 0.001 vs MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA-fibrosis but had no effect on RA-dilation. MCT increased RA-expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA-content of type-1 (proinflammatory) CD68-positive M1-macrophages without affecting type-2 (anti-inflammatory) M2-macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1-macrophages and increases in anti-inflammatory M2-macrophages versus MCT-only. MCT caused statistically-significant increases in protein-expression (Western blot) of COL3A1, ASC, CASP1, CASP8, IL1β, TGFβ3, CXCL1 and CXCL2, and decreases in MMP2, versus control. RvD1-treatment suppressed all these MCT-induced protein-expression changes.

Conclusions: The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA-remodeling, while suppressing inflammatory changes and fibrotic/electrical remodeling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodeling by suppressing ongoing inflammatory signaling.

Translational perspective: Mounting evidence suggests that chronic diseases are promoted by failure of inflammation to resolve. Here, we tested the effects of a molecule that promotes inflammation-resolution, Resolvin-D1, in a rat model of atrial arrhythmogenic remodeling caused by right-heart disease and marked by signs of persistent inflammatory signaling. Resolvin-D1 suppressed inflammatory signaling, shifted the balance of atrial macrophage-infiltration from proinflammatory M1-macrophages towards anti-inflammatory M2-macrophages and prevented atrial fibrosis. Disease-induced conduction-slowing was suppressed and atrial-fibrillation promotion was prevented. These findings support the potential value of a relatively non-toxic inflammation-resolving molecule in preventing the development of an arrhythmogenic substrate associated with chronic inflammatory signaling.

Keywords: Arrhythmia; Atrial fibrillation; Electrophysiology; Fibrosis; Inflammation; Resolvin.

Background and aim: Huashi Baidu Decoction (HSBD) is a novel complex prescription which has positive effects on severe COVID-19. This study was aimed to discover key Chinese materia medica, main active compounds, hub therapeutic target proteins and core signal pathways in the potential therapeutic mechanism of HSBD on severe COVID-19 through integrating network pharmacological methods.

Experimental procedure: TCMSP, TCMID and STITCH databases were used to screen out active compounds and target proteins of HSBD. GeneCards database was used to screen out disease genes of severe COVID-19. The potential therapeutic targets of HSBD on severe COVID-19 were used to construct protein-protein interaction network through STRING database and the hub target proteins were discovered. Next, GO and KEGG enrichment analysis were carried out to discover core signal pathways. Finally, the network diagram of "Chinese materia medica-active compounds-therapeutic target proteins" was built, then key Chinese materia medica and main active compounds were selected.

Results and conclusion: HSBD might treat severe COVID-19 through 45 potential target genes, among them, there were 13 hub target genes: RELA, TNF, IL6, IL1B, MAPK14, TP53, CXCL8, MAPK3, MAPK1, IL4, MAPK8, CASP8, STAT1. Meanswhile, GO_BiologicalProcess and KEGG signaling pathways analysis results showed that the core signal pathways were inflammation and immune regulation pathways. Finally, 4 key Chinese materia medica and 11 main active compounds were discovered in the HSBD. In conclusion, the therapeutic mechanism of HSBD on severe COVID-19 might involve its pharmacological effects of anti-inflammation and immune regulation via acting on 45 disease-related proteins of severe COVID-19.

Taxonomy classification by evise: Viral Pneumonia, COVID-19, Acute Respiratory Distress Syndrome, Septic Shock, Chinese Herbal Medicine.

Keywords: Active compounds; Anti-inflammation; Chinese materia medica; HSBD; Immune regulation.

Triple-negative breast cancer (TNBC) accounts for ~15-20% of breast cancer (BC) and has a higher rate of early relapse and mortality compared to other subtypes. The Chemokine (C-C motif) ligand 5 (CCL5) and its signaling pathway have been linked to TNBC. We aimed to investigate the susceptibility and prognostic implications of genetic variation in CCL5 signaling genes in TNBC in the present study. We characterized variants in CCL5 and that of six other CCL5 signaling genes (CCND1, ZMIZ1, CASP8, NOTCH2, MAP3K21, and HS6ST3) among 1,082 unrelated Tunisian subjects (544 BC patients, including 196 TNBC, and 538 healthy controls), assessed the association of the variants with BC-specific overall survival (OVS) and progression-free survival (PFS), and correlated CCL5 mRNA and serum levels with CCL5 genotypes. We found a highly significant association between the CCND1 rs614367-TT genotype (OR = 5.14; P = 0.004) and TNBC risk, and identified a significant association between the rs614367-T allele and decreased PFS in TNBC. A decreased risk of lymph node metastasis was associated with the MAP3K21 rs1294255-C allele, particularly in rs1294255-GC (OR = 0.47; P = 0.001). CCL5 variants (rs2107538 and rs2280789) were linked to CCL5 serum and mRNA levels. In the TCGA TNBC/Basal-like cohort the MAP3K21 rs1294255-G allele was associated with a decreased OVS. High expression of CCL5 in breast tumors was significantly associated with an increased OVS in all BC patients, but particularly in TNBC/Basal-like patients. In conclusion, genetic variation in CCL5 signaling genes may predict not only TNBC risk but also disease aggressiveness.

Lung development follows a complex series of dynamic histogenic events that depend on the fluctuation of gene expression, and the disruption of gene regulation could lead to devastating consequences, such as diseases in adulthood. In order to investigate the mechanism of lung development, we performed RNA sequencing by Illumina HiSeq™ 2000 to measure mRNA expression in lung tissues of nine rhesus macaques spanning from foetuses at gestation of 45 days to postnatal at 7 days. This development period was divided into three developmental stages, including the early stage (45-100 gestational days), the middle stage (137-163 gestational days) and the late stage (after birth at 4-7 days). Firstly, we identified stage-specific genes, based on which we found that the principle biological processes of the early stage were mainly associated with internal growth signalling, while the middle and late stage-specific genes controlled the external stress signalling. Then, we constructed a stage-specific protein-protein interaction (PPI) subnetwork, extracted dynamic modules, and identified crosstalk between modules. Moreover, we found four active pathways that could mediate the crosstalk, including the Notch signalling pathway, cell cycle, NOD-like receptor signalling pathway, and Toll-like receptor signalling pathway. These pathways not only played crucial roles in lung development, but also were implicated in lung diseases. Finally, some important bridgers, such as PSEN2, HSP90AA1 and CASP8, were discovered to explain the potential mechanism of crosstalk. Therefore, our study presents the landscape of gene expression of lung development of rhesus macaques, and provides an extended insight into the lung development mechanism.

BACKGROUND

Apoptosis plays an important role in the mechanism regulating the development of glomerulonephritis. We investigated whether polymorphisms of apoptotic genes such as B-cell CLL/lymphoma 2 (BCL2), BH3-interacting domain death agonist (BID), and caspase 8 (CASP8) were associated with immunoglobulin A nephropathy (IgAN) and with the clinical phenotypes of IgAN patients.

METHODS

We genotyped promoter and coding region single nucleotide polymorphisms (SNPs) (rs2279115 and rs1801018 for BCL2; rs8190315 and rs2072392 for BID; and rs6747918 and rs1045487 for CASP8) using direct sequencing in 195 IgAN patients and 289 control subjects.

RESULTS

No SNPs were associated with IgAN. However, in analysis of clinical phenotypes, we found that rs8190315 and rs2072392 of BID were associated with proteinuria levels of IgAN patients in additive (AG vs. GG vs. AA, p = 0.0008 for rs8190315; TC vs. CC vs. TT, p = 0.0012 for rs2072392) and dominant models (AG/GG vs. AA, p = 0.0014 for rs8190315; TC/CC vs. TT, p = 0.0031 for rs2072392). In particular, the frequencies of genotypes containing minor alleles of rs8190315 (G allele) and rs2072392 (C allele) were increased in IgAN patients with higher protienuria levels >> 40 mg/m(2)/h).

CONCLUSIONS

These results suggest that BID may play a role in severe IgAN.

There is increased interest in using cell penetrating peptides such as HIV Tat-derived peptide (Tat) to deliver biologically active cargo to cells. However, little is known about the precise molecular mechanism in cells after the uptake of Tat and after it conjugates with the cargo. Using high-density microarray analysis, we evaluated the alteration of whole genomic responses in U-937 macrophages that had been treated with Tat and Tat-fluorescein-5-isothiocynate (FITC), which served as model cargo, and then incubated for 24 h. Sixteen genes were upregulated and 28 downregulated in Tat-treated cells. After FITC had conjugated to Tat, 13 genes were upregulated and 23 downregulated. Ten upregulated and 13 downregulated genes were overlapped by both Tat and Tat-FITC. The molecular functions of regulated genes affected by Tat and Tat-FITC are described. A real-time quantitative reverse transcriptase-polymerase chain reaction was used to confirm three regulated genes (IFNAR2, CASP8, and CRLF1) affected by both Tat and Tat-FITC. We demonstrated that regulating the three genes was time-dependent and that CASP8 is also kinetically regulated in HeLa cells. Understanding the influences and consequences of Tat (Tat-cargo)-induced gene changes should help us design and develop efficient and safe delivery systems into target cells.

The expression of mRNA of genes involved in neurogenesis and apoptosis (Apaf1, Ascl1, Bax, Bcl2, Casp3, Casp8, Casp9, Dffb, Myh10, Naip2, Napa, Notch2, Numb, Pura, S100a6, Tnf) in the prefrontal cortex, hippocampus, and cerebellum was studied in adult rats. The content of mRNA of these genes (except Apaf1) was several-fold higher in the cerebellum than in the hippocampus and brain cortex. In the hippocampus, the expression of Apaf1 was significantly lower than in the prefrontal cortex, while the expression of Ascl1, Pura, S100b, and Tnf was higher. Regional differences in the direction, strength, and numbers of significant correlations between the expression of the studied genes were detected. Documented differences in gene expression were regarded as validation of the structural and functional cooperation of neurogenesis and apoptosis at the molecular genetic level.

An important unsolved problem in molecular and structural biology is the protein folding and structure prediction problem. One major bottleneck for solving this is the lack of an accurate energy to discriminate near-native conformations against other possible conformations. Here we have developed sDFIRE energy function, which is an optimized linear combination of DFIRE (the Distance-scaled Finite Ideal gas Reference state based Energy), the orientation dependent (polar-polar and polar-nonpolar) statistical potentials, and the matching scores between predicted and model structural properties including predicted main-chain torsion angles and solvent accessible surface area. The weights for these scoring terms are optimized by three widely used decoy sets consisting of a total of 134 proteins. Independent tests on CASP8 and CASP9 decoy sets indicate that sDFIRE outperforms other state-of-the-art energy functions in selecting near native structures and in the Pearson's correlation coefficient between the energy score and structural accuracy of the model (measured by TM-score).

Several descriptors of protein structure at the sequence and residue levels have been recently proposed. They are widely adopted in the analysis and prediction of structural and functional characteristics of proteins. Numerous in silico methods have been developed for sequence-based prediction of these descriptors. However, many of them do not have a public web-server and only a few integrate multiple descriptors to improve the predictions. We introduce iFC² (integrated prediction of fold, class, and content) server that is the first to integrate three modern predictors of sequence-level descriptors. They concern fold type (PFRES), structural class (SCEC), and secondary structure content (PSSC-core). The server exploits relations between the three descriptors to implement a cross-evaluation procedure that improves over the predictions of the individual methods. The iFC² annotates fold and class predictions as potentially correct/incorrect. When tested on datasets with low-similarity chains, for the fold prediction iFC² labels 82% of the PFRES predictions as correct and the accuracy of these predictions equals 72%. The accuracy of the remaining 28% of the PFRES predictions equals 38%. Similarly, our server assigns correct labels for over 79% of SCEC predictions, which are shown to be 98% accurate, while the remaining SCEC predictions are only 15% accurate. These results are shown to be competitive when contrasted against recent relevant web-servers. Predictions on CASP8 targets show that the content predicted by iFC² is competitive when compared with the content computed from the tertiary structures predicted by three best-performing methods in CASP8. The iFC² server is available at http://biomine.ece.ualberta.ca/1D/1D.html .