Prostate zones and tumor morphological parameters on magnetic resonance imaging for predicting the tumor-stage diagnosis of prostate cancer.

PURPOSE: To determine whether the morphological parameters of prostate zones and tumors on magnetic resonance imaging (MRI) can predict the tumor-stage (T-stage) of prostate cancer (PCa) and establish an optimal T-stage diagnosis protocol based on three-dimensional reconstruction and quantization after image segmentation. METHODS: A dataset of the prostate MRI scans and clinical data of 175 patients who underwent biopsy and had pathologically proven PCa from January 2018 to November 2020 was retrospectively analyzed. The authors manually segmented and measured the volume, major axis, and cross-sectional area of the peripheral zone (PZ), transition zone, central zone (CZ), anterior fibromuscular stroma, and tumor. The differences were evaluated by the One-Way analysis of variance, Pearson's chi-squared test, or independent samples t-test. Spearman's correlation coefficient and receiver operating characteristic curve analyses were also performed. The cut-off values of the T-stage diagnosis were generated using Youden's J index. RESULTS: The prostate volume (PV), PZ volume (PZV), CZ volume, tumor's major axis (TA), tumor volume (TV), and volume ratio of the TV and PV were significantly different among stages T1 to T4. The cut-off values of the PV, PZV, CZV, TA, TV, and the ratio of TV/PV for the discrimination of the T1 and T2 stages were 53.63 cm3, 11.60 cm3, 1.97 cm3, 2.30 mm, 0.90 cm3, and 0.03 [area under the curves (AUCs): 0.628, 0.658, 0.610, 0.689, 0.724, and 0.764], respectively. The cut-off values of the TA, TV, and the ratio of TV/PV for the discrimination of the T2 and T3 stages were 2.80 mm, 8.29 cm3, and 0.12 (AUCs: 0.769, 0.702, and 0.688), respectively. The cut-off values of the TA, TV, and the ratio of TV/PV for the discrimination of the T3 and T4 stages were 4.17 mm, 18.71 cm3, and 0.22 (AUCs: 0.674, 0.709, and 0.729), respectively. CONCLUSION: The morphological parameters of the prostate zones and tumors on the MRIs are simple and valuable diagnostic factors for predicting the T-stage of patients with PCa, which can help make accurate diagnoses and lateral treatment decisions.

Could tumour volume and major and minor axis based on CTA statistical anatomy improve the pre-operative T-stage in oesophageal cancer?

OBJECTIVES: To statistically study the 3D shape of oesophageal cancer (EC) and its spatial relationships based on computed tomography angiography (CTA) 3D reconstruction, to determine its relationship with T-stages, and to create an optimal T-stage diagnosis protocol based on CTA calculation. METHODS: Pre-operative CTA images of 155 patients with EC were retrospectively collected and divided into four groups: T1-T4. We used Amira software to segment and 3D reconstruct the EC, oesophagus, aorta, pericardium and peripheral lymph nodes and measured their surface area, volume, major axis, minor axis, longitudinal length, roughness and relationship to the aorta of the EC. One-way ANOVA, independent sample t-test, ROC, etc., were performed and critical values between different T-stages were calculated. We also invited two radiologists to evaluate the measurements. RESULTS: There were no significant differences in EC longitudinal length, roughness score and relationship with the aorta between the different T-stages of EC. There were significant differences in EC surface area, EC volume and mean major and minor axis among the different T-stages. The volumes of the T1-T4 tumours were 12,934.36 ± 7739.25, 23,095.27 ± 14,975.67, 37,577.98 ± 36,085.64 and 58,579.25 ± 41,073.96 mm3 separately (p < 0.05), and the T1-T4 volume cut-off values were 11,712.00, 19,809.00 and 44,103.50 mm3 separately. For comparison with radiologists, the AUC value of our measurements was 0.704, which was higher than the radiologists of AUC = 0.630. CONCLUSIONS: EC volume, major and minor axis can be used as important factors for surgeons in the T-stage diagnosis of EC, which helps to improve prognosis and treatment decisions after CTA.

Analyses of the circadian clock genes expression in whole embryos and maternal major tissues of mice.

To create an organism, it is vital to assemble enough cells of the various differentiated types with the correct spatial arrangement within the embryo. Circadian clocks development is closely correlated with all cellular differentiation. However, the expression of its emergence during mammalian development are not fully understood. To determine whether embryonic development is influenced by circadian rhythm, it is necessary to observe the ontogeny of the circadian clock gene. We first measured the expression of key circadian genes in whole embryos and maternal major tissues of 25 female mice using RT-PCR and immunohistochemical analysis. Our results indicated that mouse embryos begin to express key circadian genes and have the capacity to express active circadian regulatory cycles during development. But circadian molecular rhythms can't be built in embryo. At E15, the expression of Bmal1, Clock and Per1 mRNA in whole embryo were increased, especially Per1. In the meanwhile, immunohistochemical analysis shows a small number of PER1 positive cells were observed in the bottom of right atrium. From E16 to E17, CLOCK and PER1 positive cells were observed in the airway smooth muscle, the wall of left atrium and skeletal muscle of body wall. It is interesting that CLOCK and PER1 positive cells could not be detected in the liver. By using RT-PCR, we continue to observe the expression of myogenic regulatory factor in embryos and also analyse the relationship of embryo development and maternal rhythms. From E12, the expression of myogenin increased quickly. The expression of Tcap at E15 significantly increased. myogenin may play a direct role in contributing Tcap expression. The expression of MAZ is always the highest than myogenin and Tcap in embryos. MAZ may concern with the development of skeletal muscle. The clock gene is a positive regulator of myogenesis and the development of organ. In contrast to embryonic tissues, circadian variation was present for Bmal1, Clock and Per1 at maternal tissues. Our results indicate that circadian clock genes seem to function differently in different tissues of embryo and maternal mice. Synchrony does not occur during embryo development despite exposure to maternal rhythms. But development of embryo may be affected by maternal tissues of mice.

The interrelationship between inflammatory cytokines and skeletal muscle decay from the viewpoint of circadian rhythms.

Circadian rhythms affect a variety of physiological processes. Disruption of circadian rhythms causes many diseases, most of which are associated with inflammation. Disruption of circadian rhythms has a detrimental impact on the function of immune system. It is common to find that circulatory LPS are increased. LPS induces immune cells to produce inflammatory cytokines. Inflammatory cytokines play a role in skeletal muscle decay. Rev-erbß has been identified as a critical regulator of circadian rhythms and a factor in inflammation. Another effect of disruption is a concomitant disturbance of glucose-insulin metabolism, which skeletal muscle likely contributes to considering it is a key metabolic tissue. Disruption of circadian rhythms is also related to obesity. Obesity can cause an increase expression of inflammatory cytokines. Maybe obesity with skeletal muscle decay is one of major characteristics. Future studies are needed to obtain a comprehensive understanding of inflammatory cytokines and skeletal muscle decay from the viewpoint of circadian rhythms.

The expression of diacylglycerol kinase isoforms α and ζ correlates with the progression of experimental autoimmune encephalomyelitis in rats.

Multiple sclerosis (MS) is characterized by neuroinflammation and neurodegeneration, whose precise processes are not fully understood. Diacylglycerol kinase (DGK) isozymes of α, ß, γ and ζ expressed abundantly in the brain and/or the immune system, may be regulatory targets for MS. In this study, we analyzed the four DGK isozymes along the induction, peak and recovery phases in an experimental autoimmune encephalomyelitis (EAE) rat model of MS. The expression of these DGK isozymes and the diacylglycerol (DAG) pathway in the EAE rat brainstems were analyzed by qRT-PCR, immunohistochemistry, immunofluorescence double staining, western blotting and ELISA. Our results showed that the mRNA content of the four DGK isozymes decreased significantly, and their immunoreactivity in myelin sheathes (DGKα, ß) and neurons (DGKγ, ζ) became weaker at the beginning of the induction phase. With the progressive increase in clinical signs, DGKα, DGKγ and DGKζ mRNA increased and DGKß mRNA decreased, and microglia were involved in the formation of perivascular cuffing. In the peak phase, both DGKα and DGKζ were expressed in neurons and inflammatory cells, and DGKζ was also positive in microglia. During the recovery phase, the mRNA content and immunoreactivity of these DGK isozymes generally reached normal levels. Moreover, our results revealed that changes in DAG accumulation and PKCδ phosphorylation were almost the same as those of DGKα and DGKζ mRNA. In summary, the four DGK isozymes are involved in the EAE process. The predominant and broad presence of DGKα and DGKζ suggests that they may regulate the pathological process by attenuating DAG/PKCδ pathway signaling during EAE evolution.

Septation of the Intrapericardial Arterial Trunks in the Early Human Embryonic Heart.

BACKGROUND: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. METHODS: Serial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain. RESULTS: At CS10-CS11, the OFT wall was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT. CONCLUSIONS: Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopulmonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.

Phosphorylation of phosphatase and tensin homolog induced by Helicobacter pylori promotes cell invasion by activation of focal adhesion kinase.

Phosphorylation of the phosphatase and tensin homolog (PTEN) tumor suppressor at Ser380/Thr382/Thr383 residues is a novel mechanism underlying PTEN inactivation in gastric carcinogenesis, which may be triggered by Helicobacter pylori infection. To investigate this further, the effect of H. pylori infection on PTEN phosphorylation and the subsequent activation of focal adhesion kinase (FAK), were evaluated in gastric tissue samples from Mongolian gerbils and in the human gastric epithelial mucosa cell line GES-1 using immunohistochemistry, western blotting and Transwell assays. The in vivo and in vitro results of the present study demonstrated that H. pylori infection induced the phosphorylation and inactivation of PTEN at Ser380/Thr382/383, and the subsequent phosphorylation and activation of FAK at Tyr397. Gastric epithelial cell invasion was also increased. Furthermore, stable expression of a dominant-negative PTEN mutant inhibited the enhanced FAK activation and cell invasion induced by H. pylori infection. These results suggest that the mechanism underlying H. pylori-induced carcinogenesis may involve promoting cell invasion through the phosphorylation of PTEN and the activation of FAK.

Expression of c-Met and hepatocyte growth factor in various gastric pathologies and its association with Helicobacter pylori infection.

Activation of the hepatocyte growth factor (HGF)/c-Met signaling pathway was identified to be associated with malignant tumors. The present study aimed at determining the expression of HGF and c-Met in gastric carcinogenesis and its association with Helicobacter pylori infection. Gastric biopsies were obtained from 160 H. pylori-negative and -positive patients, including those with chronic gastritis, intestinal metaplasia, dysplasia and gastric cancer (GC). Proteins were extracted from GES-1, gastric epithelial, and AGS, gastric adenocarcinoma, cells following co-culture with H. pylori in vitro. The expression of HGF and c-Met in gastric tissues or cells was determined using immunohistochemistry and western blot analysis. The expression of c-Met increased in GC tissues (72.5%) compared with that in pre-cancerous lesions (17.5, 17.5 and 30%). Additional analysis identified that the expression of HGF and c-Met was significantly increased in the presence of H. pylori infection in dysplasia and gastric cancer samples. Furthermore, H. pylori may activate the HGF/c-Met signaling pathway in vitro, which may contribute to gastric carcinogenesis.

Phosphorylation and inactivation of PTEN at residues Ser380/Thr382/383 induced by Helicobacter pylori promotes gastric epithelial cell survival through PI3K/Akt pathway.

Phosphorylation of PTEN at residues Ser380/Thr382/383 leads to loss of phosphatase activity and tumor suppressor function. Here, we found that phosphorylation of PTEN at residues Ser380/Thr382/383 was increased with gastric carcinogenesis, and more importantly, Helicobacter pylori was a trigger of this modification in chronic non-atrophic gastritis. H. pylori could phosphorylate and inactivate PTEN in vivo and in vitro, resulting in survival of gastric epithelial cells. Furthermore, stable expression of dominant-negative mutant PTEN or inhibition of Akt prevented the enhanced survival induced by H. pylori. These results indicate that PTEN phosphorylation at residues Ser380/Thr382/383 is a novel mechanism of PTEN inactivation in gastric carcinogenesis, and H. pylori triggers this modification, resulting in activation of the PI3K/Akt pathway and promotion of cell survival.

Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification.

Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.

Expression of γH2AX in various gastric pathologies and its association with Helicobacter pylori infection.

Phosphorylation of H2AX at Ser 139 (γH2AX) is a biomarker of DNA double-strand breaks (DSBs). The present study aimed to explore the association between γH2AX levels and gastric pathology and Helicobacter pylori (H. pylori) infection. Gastric biopsies were obtained from 302 H. pylori-negative and -positive patients, including those with chronic gastritis (CG), intestinal metaplasia (IM), dysplasia (Dys) and gastric cancer (GC). Proteins were extracted from five gastric epithelial cell lines and from 10 specimens of matched GC and adjacent normal tissues. The expression of γH2AX, a biomarker for the detection of DNA DSBs, in gastric tissues was detected by immunohistochemistry and western blotting. The expression of γH2AX progressively increased in tissues according to pathological stage from CG to Dys, but was slightly decreased in GC. H. pylori infection was associated with increased γH2AX expression, IM and Dys. Overexpression of γH2AX in GC was found to correlate with tumor location, gross appearance, differentiation, depth of invasion, TNM stage and lymph node metastasis. The results indicated that DSBs appear to be an early molecular event in gastric carcinogenesis, which may be associated with H. pylori infection. Moreover, immunohistochemical staining of γH2AX was found to correlate with a number of clinicopathological characteristics. The expression of γH2AX may serve as a valuable biomarker for the diagnosis and progression of GC.

Second heart field and the development of the outflow tract in human embryonic heart.

The second heart field (SHF) is indicated to contribute to the embryonic heart development. However, less knowledge is available about SHF development of human embryo due to the difficulty of collecting embryos. In this study, serial sections of human embryos from Carnegie stage 10 (CS10) to CS16 were stained with antibodies against Islet-1 (Isl-1), Nkx2.5, GATA4, myosin heavy chain (MHC) and α-smooth muscle actin (α-SMA) to observe spatiotemporal distribution of SHF and its contribution to the development of the arterial pole of cardiac tube. Our findings suggest that during CS10 to CS12, SHF of the human embryo is composed of the bilateral pharyngeal mesenchyme, the central mesenchyme of the branchial arch and splanchnic mesoderm of the pericardial cavity dorsal wall. With development, SHF translocates and consists of ventral pharyngeal mesenchyme and dorsal wall of the pericardial cavity. Hence, the SHF of human embryo shows a dynamic spatiotemporal distribution pattern. The formation of the Isl-1 positive condense cell prongs provides an explanation for the saddle structure formation at the distal pole of the outflow tract. In human embryo, the Isl-1 positive cells of SHF may contribute to the formation of myocardial outflow tract (OFT) and the septum during different development stages.

Critical selection of internal control genes for quantitative real-time RT-PCR studies in lipopolysaccharide-stimulated human THP-1 and K562 cells.

The choice of internal control genes is important since it may affect the study outcome in RT-qPCR. Indeed, it is well-known that expression levels of traditional internal control genes can vary across tissue types and across experimental settings within one specific tissue type. The aim of this study is an evaluation of a set of housekeeping genes (HKGs) to be used in the normalization of gene expression in vitro different cultured cells, THP-1 and K562. The transcriptional stability of eleven potential internal control genes (RPL37A, ACTB, GAPDH, B(2)M, PPIB, PGK1, PPIA, SDHA, TBP, HPRT1 and RPL13A) were evaluated using RT-qPCR and were compared in different treatment, that was un-stimulated or LPS-stimulated cells. The raw Ct values were determined for each candidate gene at different time points following LPS-stimulated or unstimulated cells. Furthermore, all data were analyzed by the geNorm, BestKeeper, and NormFinder validation programs. Results indicated that PPIB and PGK1 were the most stable internal control genes in this study. RPL13A was found to be the least stable. This study provides the comprehensive reported assessment of internal control genes for use in expression studies in vitro cultured cells. These findings further emphasize the need to accurately validate candidate internal control genes in the study before use in gene expression studies using RT-qPCR.

Paraventricular nucleus corticotrophin releasing hormone contributes to sympathoexcitation via interaction with neurotransmitters in heart failure.

Recent studies indicate that systemic administration of tumor necrosis factor (TNF)-α induces increases in corticotrophin releasing hormone (CRH) and CRH type 1 receptors in the hypothalamic paraventricular nucleus (PVN). In this study, we explored the hypothesis that CRH in the PVN contributes to sympathoexcitation via interaction with neurotransmitters in heart failure (HF). Sprague-Dawley rats with HF or sham-operated controls (SHAM) were treated for 4 weeks with a continuous bilateral PVN infusion of the selective CRH-R1 antagonist NBI-27914 or vehicle. Rats with HF had higher levels of glutamate, norepinephrine (NE) and tyrosine hydroxylase (TH), and lower levels of gamma-aminobutyric acid (GABA) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN when compared to SHAM rats. Plasma levels of cytokines, NE, ACTH and renal sympathetic nerve activity (RSNA) were increased in HF rats. Bilateral PVN infusions of NBI-27914 attenuated the decreases in PVN GABA and GAD67, and the increases in RSNA, ACTH and PVN glutamate, NE and TH observed in HF rats. These findings suggest that CRH in the PVN modulates neurotransmitters and contributes to sympathoexcitation in rats with ischemia-induced HF.