The modulation relationship of genomic pattern of intratumor heterogeneity and immunity microenvironment heterogeneity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, with the second highest mortality rate among all cancer types. Growing evidence has demonstrated the notable effects of intratumor heterogeneity (ITH) and tumor immune microenvironment heterogeneity (TIMH) on the biological processes involved in HCC. However, the interactive mechanisms between ITH and TIMH is still unclear. The present study systematically screened the mRNA expression, simple nucleotide variation data and clinical data of samples from The Cancer Genome Atlas (TCGA). The mutant-allele tumor heterogeneity (MATH) score was used to represent ITH, and TCGA cohort was divided into two groups according to the MATH score. Next, different immune-related signaling pathways and enriched immune-related genes were identified using Gene Set Enrichment Analysis of these two groups, and the results revealed that interleukin-1α (IL1A) and serine/threonine-protein kinase PAK4 were associated with prognosis. Furthermore, CIBERSORT was utilized to calculate the fractions of 22 types of leukocytes to represent TIMH, and the fractions of M1 and M2 macrophages were confirmed to be associated with prognosis. Therefore, PAK4, interleukin-1α (IL1A), and M1/M2 ratio were selected as the key factors involved in the interaction between ITH and TIMH. Afterwards, microRNAs (miRNAs) that were linearly related to the M1/M2 ratio and the potential target genes of the miRNAs were screened. Finally, the regulatory network between PAK4, IL1A, and the M1/M2 ratio was established, bridged by the above miRNAs and the target genes. In addition, PAK4, heat shock protein 105 kDa and miRNA-1911 were demonstrated to be a key factor involved in immune response via Weighted Correlation Network Analysis in HCC.

Identification of phosphorylated MYL12B as a potential plasma biomarker for septic acute kidney injury using a quantitative proteomic approach.

Acute kidney injury (AKI) is a common and increasingly encountered complication in hospitalized patients with critical illness in intensive care units (ICU). According to the etiology, Sepsis-induced AKI (SAKI) is a leading contributor to AKI and significantly has very poor prognosis, which might be related to the late detection when the elevation of BUN and serum creatinine (SCr) is used. Many genes are up-regulated in the damaged kidney with the corresponding protein products appearing in plasma and urine. Some of these are candidate biomarkers for more timely diagnosis of SAKI. Therefore, extensive research efforts over this past decade have been directed at the discovery and validation of novel SAKI biomarkers to detect injury prior to changes in kidney function, a number of serum and urinary proteins, including NGAL, KIM-1, cystatin-C, IL-18, and L-FABP, have been identified for predicting SAKI before a rise in BUN and serum creatinine in several experimental and clinical trainings. Unfortunately, an ideal biomarker of SAKI with highly sensitivity and specificity has not been identified yet. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for SAKI. In the present study, kidney tissue samples from SAKI mice were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and 4 up-regulated proteins, which were actin (ACTB), myosin regulatory light chain 12B (MYL12B), myosin regulatory light polypeptide 9 (MYL9), and myosin regulatory light chain 12A (MYL12A) were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Among all the varied proteins, MYL12B was validated by western blot. Interestingly, there was no change between the SAKI and control kidney tissues, however, phosphorylated MYL12B was detected to be consistent with the proteomics data. Furthermore, phosphorylated MYL12B was found similarly to be increased in SAKI plasma, while MYL12B was changeless in plasma of control group. Taking together, phosphorylated MYL12B may be employed as a potential plasma biomarker for the early diagnosis of SAKI.

Blockage of protease-activated receptor 1 ameliorates heat-stress induced intestinal high permeability and bacterial translocation.

Accumulated evidences indicate intestinal lesions play an important role in the pathogenesis of heatstroke. However, the underlying mechanisms by which heat stress causes intestinal barrier dysfunction and bacterial translocation remain unclear. In this study, we investigated the role of protease-activated receptor 1 (PAR1) in heat stress-induced intestinal hyper-permeability and bacterial translocation. Intestinal permeability in heat stressed mouse was evaluated by determining plasma endotoxin concentration and urinal lactulose/mannitol (L/M) ratio with gastric administration of L/M solution. Venous blood, liver, spleen and mesenteric lymph node tissues were collected for bacterial load test. Real time PCR was used to determine ileum PAR1 mRNA expression. In vitro study, permeability was assessed by determining trans-epithelial electrical resistance (TEER) in human intestinal Caco-2 cell line. RWJ-58259, a selective antagonist of PAR1, was used both in vivo and in vitro studies. The results showed that heat stress could increase ileum PAR1 mRNA level, urinal L/M ratio, plasma endotoxin concentration and bacterial load in the blood, spleen and mesenteric lymph nodes. Blocking PAR1 with RWJ-58259 (10 mg/kg) pretreatment could significantly reduce heat stress-induced above changes, but have no role to PAR1 mRNA level. In Caco-2 cells, heat stress-induced high permeability could also be reduced by RWJ-58259 (5-20 µmol/L). In summary, our results demonstrated that PAR1 signaling pathway may play an important role in the heat stress-induced elevation of intestinal permeability, bacterial translocation and the occurrence of endotoxemia.

Pathological changes in the lung and brain of mice during heat stress and cooling treatment.

BACKGROUND: Heatstroke often leads to multiple organ dysfunction syndrome (MODS) with a death rate of 40% or a neurological morbidity of 30%. These high rates in patients with heatstroke are largely due to the progression of heat stress to MODS, resulting in no specific treatment available. This study aimed to develop a mouse model of heat stress and determine the pathological changes in the lung and brain during heat stress and cooling treatment. METHODS: A mouse model of heat stress was established in a pre-warmed incubator set at 35.5 ± 0.5°C and with a relative humidity of 60% ± 5%. Rectal temperature was monitored, and at a temperature of 39 °C, 40 °C, 41 °C, or 42 °C, the mice were sacrificed. The remaining animals were removed from the incubator and cooled at an ambient temperature of 25 ± 0.5 °C and a humidity of 35% ± 5% for 12 or 24 hours at a temperature of 41 °C or for 6 hours at a temperature of 42 °C. The control mice were sham-heated at a temperature of 25 ± 0.5 °C and a humidity of 35% ± 5%. The lungs and brains of all animals were isolated. Hematoxylin and eosin staining and light microscopy were performed to detect pathological changes. RESULTS: All mice demonstrated a uniform response to heat stress. A low degree of heat stress induced marked pathological changes of the lungs. With the rise of the temperature to 42°C, progressively greater damage to the lungs with further congestion of the lung matrix, asystematic hemorrhage of alveolar space, abscission of alveolar epithelial cells, and disappearance of pulmonary alveolus tissue structure were detected. However, absorption of congestion and hemorrhage as well as recovery of pulmonary alveolus tissue structure was observed following cooling treatment at an ambient temperature. With a low degree of heat stress, the brain only showed moderate edema. Neuronal denaturation and necrosis were detected at a temperature of 42°C. Interestingly, the lesions in the brain were further aggravated at 42 °C regardless of cooling treatment, but recovery was observed after cooling treatment at 41 °C. CONCLUSIONS: The pathological changes of the lungs and brain of mice showed distinctive lesions following heat stress and cooling treatment, and they were correlated with the time and duration of cooling treatment. The results of this study are helpful for further study of the mechanisms linking heatstroke.

The effects of different sample labelling methods on signal intensities of a 60-mer diagnostic microarray.

The effects of four different labelling methods on signal intensities of a 60-mer diagnostic microarray were studied. Eighty of virus-specific oligonucleotide probes for human influenza virus were prepared in an array of 15x16 spots. RNA samples from cultured human influenza virus strains were labelled with four different methods, including direct cDNA labelling (DL), universal primer labelling (UPL), direct cDNA labelling with restriction display (DL-RD), and Cy-dUTP incorporated cDNA labelling with restriction display (IL-RD) in a signal color format. The background-subtracted signal intensities from five replicate hybridization experiments of each labelling method were analyzed using one-way analysis of variance (one-way ANOVA) and linear regression techniques. The effect of sample labelling method on background-subtracted signal intensities was significant (p<0.001) and multiple comparisons showed the differences existed mainly between DL and the other three labelling methods. The sample labelling method explained about 4.3% of signal intensity. The results demonstrated that UPL and the RD-based methods are more efficient than the conventional DL method for sample labelling, an important variation factor affecting the signal intensities in diagnostic microarrays.

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

[A new fluorescent labeling technique in microarray studies: universal primer U2 labeling].

OBJECTIVE: To develop a new method for fluorescent labeling technique, universal primer U(2) labeling (UPL), for microarray studies. METHOD: Influenza virus RNA was labeled with four labeling methods, namely UPL, random primer, restriction display incorporation labeling method and reverse transcription coupled random primer spiking labeling method (RT-PSL), respectively, and hybridized to influenza virus oligonucleotide microarray. The signals extracted from the microarrays were analyzed with SPSS 10.0 software to compare the efficiency and reproducibility of the labeling methods. RESULTS: The fluorescence intensity, signal-to-noise ratio (SNR), true positive ratio (TPR) of the probes and reproducibility of labeling with UPL were comparable with those RD-labeling method, and higher than those of RT-PSL method. UPL reduced the complexity of the procedures in comparison with the other labeling methods. CONCLUSION: UPL labeling method can be used in research and development of the microarray technique.

[Oligonucleotide microarray for human immunodeficiency virus detection].

OBJECTIVE: To develop an oligonucleotide microarray for fast detection of human immunodeficiency virus (HIV). METHODS: With complete genome sequence of HIV-1 subtype B (U26942) as the target sequence and bioinformatics software such as DNAClub, Oligo6.0, BLAST, Alignment, oligonucleotide probes of high specificity with identical length and similar melting temperature (T(m)) were designed and synthesized. Oligonucleotide microarray was prepared using Cartesian Microarrayer. Using the plasmids of HIV-1 subtype B(U26942), C(U46016), F(AF075703), G(AF061640) and restriction display technique, Cy3-labeled HIV DNA fragments were amplified and the hybridization results were scanned and analyzed with Array-Pro. RESULTS AND CONCLUSION: Twenty-two optimized oligonucleotide microarray probes were obtained and used to prepare the oligonucleotide microarray for further screening studies. The microarray prepared significantly enhanced the sensitivity, reliability and speed of DNA assay, and possesses the potential for application in clinical setting.