Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis

AIM: To determine the role of heparanase-1 (HPSE-1) in orbital rhabdomyosarcoma (RMS), and to investigate the feasibility of HPSE-1 targeted therapy for RMS. METHODS: Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients (including 28 cases of embryonal RMS and 23 cases of alveolar RMS), among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy. In vitro, studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). RD cells (an RMS cell line) and HUVECs were infected with HPSE-1 shRNA lentivirus at a multiplicity of infection (MOI) of 10 and 30 separately. Real-time PCR and Western blot were applied to detect the mRNA and protein expression levels of HPSE-1. Cell viability of treated or control RD cells was evaluated by cell counting kit-8 (CCK-8) assay. Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs. RESULTS: Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9% in orbital embryonal RMS and 91.3% in orbital alveolar RMS. Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS. However, despite the types of RMS, comparing the cases treated chemoradiotherapy with those untreated, we have observed that chemoradiotherapy resulted in weaker staining in patients' tissues. The expression levels of HPSE-1 declined significantly in both the mRNA and protein levels in HPSE-1 shRNA transfected RD cells. The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro. Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel. CONCLUSION: HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.

Analysis of intronless genes involved in oscillation and differentiation

The genomes of higher eukaryotes are replete with intron-containing genes. Transcription of these genes produces precursor mRNAs containing intervening sequences, which are subsequently removed and the exons spliced together to form the mature mRNA. However, a small proportion of eukaryotic protein-coding genes are intronless and therefore bypass post-transcriptional splicing events. Although a large proportion of intronless genes are known to code for certain types of proteins, their specific role in the genome of higher organism is perplexing. This research set out to elucidate the functions of intronless genes in humans by studying their involvement in the expression pattern of oscillatory gene that occurs in the pre-somitic mesoderm of developing embryo. Twenty-seven (27) human homologs of mouse oscillatory genes were analysed to determine the number of exons present in them using various bioinformatics databases. The result obtained identified two intronless genes –NRARP and ID1 – which are associated with the Notch signalling pathway of the segmentation clock. This represented 7.4% of the total oscillatory genes analysed. No intronless gene was found in the Wnt and FGF signalling pathways – two other pathways famous for oscillatory gene expression. The proteins encoded by the intronless genes are involved in several important biological processes including angiogenesis, cell cycle control and in the regulation of cellular senescence. Although oscillatory genes had fewer numbers of introns compared to the non-oscillatory genes, the intronless genes were not implicated in the regulation of the precise timing events of the segmentation clock. This result may also point to the fact that the rapid expression rate of the oscillatory genes in the PSM may favour the reduced intron length of the oscillatory genes.

Segnet Network Algorithm-Based Ultrasound Images in the Diagnosis of Gallbladder Stones Complicated with Gallbladder Carcinoma and the Relationship between P16 Expression with Gallbladder Carcinoma

The study focused on how to improve the diagnostic coincidence rate of patients with gallbladder stones and gallbladder cancer based on an optimized Segnet network algorithm and the relationship of gallbladder cancer with multiple tumor suppressor 1 (P16). 300 patients diagnosed with gallbladder cancer in the hospital were selected as the research subjects. The pyramid pooling operation was incorporated into the original Segnet network algorithm, and its performance was evaluated, factoring into the intersection of union (IoU), algorithm precision (Pre), and recall rate (Recall). After 8 hours of fasting, conventional ultrasound and contrast-enhanced ultrasound examinations were performed, and the images were evaluated by three experienced ultrasound diagnosticians. The positive signal of P16 immunohistochemical staining was brownish yellow, which was generally concentrated in the nucleus, and a small part was located in the cytoplasm. In each slice, ten visual fields were selected. Then, they were observed under a high-power mirror, and the number was counted. It was found that the optimized Segnet network algorithm increased the IoU by 7.3%, the precision by 8.2%, and the recall rate by 11.1%. The diagnostic coincidence rates of conventional ultrasound and contrast-enhanced ultrasound examinations for gallbladder cancer were 78.13% (25/32) and 87.5% (25/32), respectively. The positive expression rate of P16 in gallbladder adenocarcinoma (47.06%) was significantly lower than that of acute cholecystitis with gallbladder stones (84.38%) and gallbladder polyps (67.16%) ( P < 0.05 ). The positive expression rate of P16 in patients with stage III and stage IV (33.33% and 40%) was significantly lower than that in patients with stages I and II (87.5% and 80%) ( P < 0.05 ). The positive expression rate of P16 in high differentiation (86.67%) was significantly higher than that of moderate differentiation (40%) and poor differentiation (28.57%) ( P < 0.05 ). In short, contrast-enhanced ultrasound can effectively improve the diagnostic coincidence rate of gallbladder cancer, and the expression of P16 in gallbladder cancer is closely related to tumor staging and differentiation.

Clinical Study of THSD7A, SOD2 and AR Antigens in Idiopathic Membranous Nephropathy

Background To explore the expression of THSD7A, SOD2 and AR podocyte antigens in the renal tissue of patients with idiopathic membranous nephropathy (IMN) and its clinical diagnostic value in IMN. Method This study retrospectively collected 150 renal tissue specimens and clinical data of patients undergoing renal biopsy in the Department of Nephrology, Shandong Provincial Hospital, including 130 IMN and 20 non-IMN patients. We use immunohistochemical staining to detect the expression of THSD7A, SOD2, AR antigens in the kidney tissue of patients. We focus on analyzing the clinical and pathological characteristics of IMN patients with positive THSD7A antigen expression, and analyze the prognosis of THSD7A-IMN patients and tumor occurrence. Meanwhile, the sensitivity and specificity of those antigens for the diagnosis of IMN were discussed. Results There were only 6 patients with THSD7A positive expression among 150 patients with glomerular disease, of which 5 patients were all IMN patients, and the patients' serum anti-PLA2R antibodies(anti-PLA2R-ab) were all negative. The positive rate of THSD7A in IMN was 3.85%, and the positive rate in IMN patients with anti-PLA2R-ab negative was 5.50%. Meanwhile, the sensitivity and specificity of renal tissue THSD7A in diagnosing IMN is 4.0% and 95%. In addition, the IgG subtype of THSD7A-IMN was mainly IgG4; Among them, 2 patients with IMN had both THSD7A and PLA2R antigen staining. This study also suggests that there is no significant difference in clinical and pathological characteristics between THSD7A positive and negative group. The follow-up of THSD7A-IMN patients found no tumors. This study also showed that there were 11 cases of anti-PLA2R-ab negative IMN patients with SOD2 antigen expression (8.56%), 10 cases of anti-PLA2R-ab negative and 4 cases of anti-PLA2R-ab positive IMN patients with AR antigen expression (10.77%), but no obvious SOD2 and AR antigen expression in SMN and other glomerular diseases. Conclusions Glomerular THSD7A antigen has a certain expression rate and high specificity in IMN, but its expression rate is low, and it can be used as a specific diagnostic index for IMN supplement. Kidney tissue SOD2, AR podocyte target antigens have a low positive rate in IMN, and further research is needed to evaluate the condition of IMN.

Evaluation of P53 gene expression by immunohistochemistry to diagnosis oral precancerous lesions

Oral Precancerous lesions include leukoplakia, erythroplakia, and mucosa palate changes due to reverse smoking. Assessing the prevalence of these lesions in a cross-sectional study can be effective in the timely prevention and treatment of lesions, in any community. Hence, in the present study, evaluation of P53 gene expression was done by immunohistochemistry method to diagnosis oral precancerous lesions. For this purpose, 111 Chinese patients (54 women and 57 men) were selected for examination. The age range of these patients was 22 to 69 years, and their average age was 32.6 years. All patients were examined by one physician. Oral mucosa was used for immunohistochemical evaluations. All samples taken from patients' mucosa were evaluated by one pathologist under a light microscope. 80 cases of the 111 patients were smokers and 27 were non-smokers. Among the 80 smokers, 56.25% had leukoplakia, 3.75% had erythroplakia, and 40% had mucosa palate changes. Regarding non-smokers, 74.07% had leukoplakia and 25.93% had erythroplakia. None of the non-smokers had mucosa palate changes. In terms of the lesion location, in patients with leukoplakia 89.23%, and patients with erythroplakia 90% of the lesion was located in the cheek mucosa and buccal vestibule. Also, in patients with leukoplakia 9.23%, and patients with erythroplakia 10% of the lesion was located in the lips vestibular mucosa. Only 1.54% of leukoplakia had a lesion in the vermilion border, and none of the erythroplakia patients had a lesion on the vermilion border. 76 patients (68.46%) showed positive expression of the P53 gene. The expression level of the P53 gene did not show a significant relationship with age, and the genders did not have a statistically significant difference in terms of gene expression. The expression level of the P53 gene was 59.8% in leukoplakia, 70% in erythroplakia, and 40% in Mucosa palate changes. The present study showed that the evaluation of P53 gene expression was well able to detect oral precancerous lesions and their severity by increasing their expression rate.

Unveiling the Role Displayed by Penicillium digitatum PdMut3 Transcription Factor in Pathogen–Fruit Interaction

Zn2Cys6 transcription factors are unique to fungi and are involved in different regulatory functions. In this study, we have identified the Penicillium digitatumPdMut3 gene, which encodes a putative Zn (II) 2Cys6 DNA-binding protein. Elimination of PdMut3 in Pd1 strain caused increased virulence during citrus infection. The transcription of the PdMut3 gene showed a higher expression rate during fungal growth and less transcription during fruit infection. Furthermore, the deletion of the gene in the wild-type isolate of P. digitatum did not produce any modification of the sensitivity to different fungicides, indicating that the gene is not associated with resistance to fungicides. In contrast, PdMut3 null mutants showed a reduction in growth in minimal media, which was associated with severe alterations in conidiophore development and morphological alterations of the hyphae. Mutants showed greater sensitivity to compounds that interfere with the cell wall and an invasive growth block. Thus, PdMut3 might have an indirect role in fungi virulence through metabolism and peroxisomes development.

The Restorative Effect of Human Amniotic Fluid Stem Cells on Spinal Cord Injury

Spinal cord injury (SCI) is a debilitating condition within the neural system which is clinically manifested by sensory-motor dysfunction, leading, in some cases, to neural paralysis for the rest of the patient’s life. In the current study, mesenchymal stem cells (MSCs) were isolated from the human amniotic fluid, in order to study their juxtacrine and paracrine activities. Flow cytometry analysis was performed to identify the MSCs. A conditioned medium (CM) was collected to measure the level of BDNF, IL-1β, and IL-6 proteins using the ELISA assay. Following the SCI induction, MSCs and CM were injected into the lesion site, and also CM was infused intraperitoneally in the different groups. Two weeks after SCI induction, the spinal cord samples were examined to evaluate the expression of the doublecortin (DCX) and glial fibrillary acid protein (GFAP) markers using immunofluorescence staining. The MSCs’ phenotype was confirmed upon the expression and un-expression of the related CD markers. Our results show that MSCs increased the expression level of the DCX and decreased the level of the GFAP relative to the injury group (p < 0.001). Additionally, the CM promoted the DCX expression rate (p < 0.001) and decreased the GFAP expression rate (p < 0.01) as compared with the injury group. Noteworthily, the restorative potential of the MSCs was higher than that of the CM (p < 0.01). Large-scale meta-analysis of transcriptomic data highlighted PAK5, ST8SIA3, and NRXN1 as positively coexpressed genes with DCX. These genes are involved in neuroactive ligand–receptor interaction. Overall, our data revealed that both therapeutic interventions could promote the regeneration and restoration of the damaged neural tissue by increasing the rate of neuroblasts and decreasing the astrocytes.

HER2 and p53 Expression in Marjolin’s Ulcer

Background：Marjolin's ulcer is a malignant tumor that is different from other skin ulcers, and its pathogenesis is not yet clear. The diagnosis and prognosis of Marjolin's ulcer lack valuable marks on immunohistochemistry (IHC).Methods: In this study, we detected the expression of HER2 and p53 in Majorlin’s ulcer with immunohistochemistry, and retrospectively analyze the clinicopathological characteristics of Marjolin's ulcer samples.Results: Our results showed that no HER2 but p53 was detected in Majorlin’s Ulcer samples. Meanwhile, by statistic analysis we found that the positive expression rate of p53 in Majorlin’s Ulcer samples was associated with sex, course of disease, ulcer size, pathological type of ulcer canceration, and degree of tumor differentiation. Furthermore, we showed that the positive rate of p53 was proportional to the malignancy degree of Marjolin’s ulcer. Conclusions:Our results of this study will lay a foundation for diagnosis of Marjolin's ulcer and even prevention of Marjolin's ulcer progression

NOTCH Target Gene HES5 Distinct Anti-Tumorigenic Roles in Gastric Carcinoma

I. Background:Gastric cancer (GC) is ranked the third greatest cause of mortality globally and the second common cancer in Iran. To date, many pathways including HES family have been found to be linked to cellular proliferation, differentiation and cancer. HES5, a transcription factor that binds to DNA, is known as a downstream protein in Notch pathway. Therefore, we aimed to investigate the association between HES5 expression and GC.II. Methods and Results:In this study,75 gastric cancer patients were included. After RNA extraction and cDNA synthesis, the expression rate of HES5 was evaluated by quantitative Real-time PCR. Also, presence of H. Pylori infection was verified using specific primers set for H. pylori, and PCR on extracted DNA. The results showed a remarkable decrease in HES level in malignant tissues when compared to the neighboring non-cancerous tissues (Normal) (P< 0.0001). Moreover, an inverse relation between down regulation of HES 5 gene and H.Pylori infection was identified. In conclusionIII. Conclusions: These findings emphasize that HES5 may notably suppress the tumor development.

PAOX1 expression in mixed-substrate continuous cultures of Komagataella phaffii (Pichia pastoris) is completely determined by methanol uptake regardless of the secondary carbon source: a simplifying principle for predicting recombinant protein production.

The expression of recombinant proteins by the AOX1 promoter of Komagataella phaffii is typically induced by adding methanol to the cultivation medium. Since growth on methanol imposes a high oxygen demand, the medium is often supplemented with an additional "secondary" carbon source which serves to reduce the consumption of methanol, and hence, oxygen. Early research recommended the use of glycerol as the secondary carbon source, but more recent studies recommend the use of sorbitol because glycerol represses PAOX1 expression. To assess the validity of this recommendation, we measured the steady state concentrations of biomass, residual methanol, and AOX1 over a wide range of dilution rates (0.02-0.20 h-1) in continuous cultures of the Mut+ strain fed with methanol, methanol + glycerol, and methanol + sorbitol. We find that when the specific AOX1 expression and methanol uptake rates for each of the three feeds are plotted against each other, they collapse into a single hyperbolic curve. The specific AOX1 expression rate is therefore completely determined by the specific methanol uptake rate regardless of the existence (present/absent) and type (repressing/non-repressing) of the secondary carbon source. In particular, cultures fed with methanol + glycerol and methanol + sorbitol that consume methanol at equal rates also express the protein at equal rates and levels. Now, it turns out that the simple unstructured model developed by Egli and co-workers can predict the specific methanol uptake rates of single- and mixed-substrate cultures over a wide range of dilution rates and feed concentrations. By combining this model with our data, we derive simple formulas that predicts the protein expression rates and levels of single- and mixed-substrate cultures over a wide range of conditions.