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Abstract Phase singularities are phase-indeterminate points where wave amplitudes are zero, which manifest as phase vertices or wavefront dislocations. In the realm of optical and electron beams, the phase singularity has been extensively explored, demonstrating a profound connection to orbital angular momentum. Direct local imaging of the impact of orbital angular momentum on phase singularities at the nanoscale, however, remains challenging. Here, we study the role of orbital angular momentum in phase singularities in graphene, particularly at the atomic level, through scanning tunneling microscopy and spectroscopy. Our experiments demonstrate that the scatterings between different orbital angular momentum states, which are induced by local rotational symmetry-breaking potentials, can generate additional phase singularities, and result in robust single-wavefront dislocations in real space. Our results pave the way for exploring the effects of orbital degree of freedom on quantum phases in quasiparticle interference processes.

ObjectivesDysphagia is a common complication in stroke patients, widely affecting recovery and quality of life after stroke. The objective of this systematic review is to identify the gaps that between evidence and practice by critically assessing the quality of clinical practice guidelines (CPGs) for management of dysphagia in stroke.MethodsWe systematically searched academic databases and guideline repositories between January 1, 2014, and August 1, 2023. The Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument was used by two authors to independently assess CPG quality.ResultsIn a total of 14 CPGs included, we identified that three CPGs obtained a final evaluation of “high quality,” nine CPGs achieved “moderate quality” and two CPGs received “low quality.” The domain of “scope and purpose” achieved the highest mean score (91.1%) and the highest median (IQR) of 91.7% (86.1, 94.4%), while the domain of “applicability” received the lowest mean score (55.8%) and the lowest median (IQR) of 55.4% (43.2, 75.5%).ConclusionThe CPG development group should pay more attention to improving the methodological quality according to the AGREE II instrument, especially in the domain of “applicability” and “stakeholder involvement;” and each item should be refined as much as possible.

Abstract Background Porphyromonas gingivalis plays an oncogenic role in development and progression of esophageal squamous cell carcinoma (ESCC). However, the impact of P. gingivalis on local recurrence of early ESCC or precancerous lesion after ESD treatment remains unknown. The present study aimed to evaluate the impact of P. gingivalis on local recurrence after ESD treatment of early ESCC or high-grade dysplasia (HGD). Methods The amount of P. gingivalis was assessed by immunohistochemistry in 205 patients with early ESCC or HGD. Univariate and multivariate Cox regression analyses were performed to determine the effect of P. gingivalis on local recurrence. Propensity score matching analysis was performed to reduce the imbalance of baseline characteristics. A nomogram integrating significant prognostic factors was built for local recurrence prediction. Results The amount of P. gingivalis increased significantly in neoplasms that invaded up to muscularis mucosa and submucosa compared with lesions confined to epithelium or lamina propria. Overabundance of P. gingivalis was positively associated with invasion depth, post-ESD stricture and local recurrence. Univariate and multivariate Cox regression analyses revealed that P. gingivalis, longitudinal length of lesion and lymphovascular invasion were independent predictors for post-ESD recurrence. A nomogram comprising P. gingivalis, lymphovascular involvement, and lesion length performed well for prediction of post-ESD local recurrence with the concordance indices of 0.72 (95%CI, 0.62 to 0.80), 0.72 (95%CI, 0.63 to 0.80), and 0.74 (95%CI, 0.65 to 0.83) in the validation cohort, the entire cohort, and the subcohort after PSM, respectively. Conclusion P. gingivalis overabundance is a risk factor and a potential predictor for local recurrence of early ESCC or HGD after ESD treatment. Thus, clearance of P. gingivalis represents an attractive strategy for prognosis improvement and for prevention of ESCC.

WNT inhibitory factor 1 (WIF1) is known to function as a tumor suppressor gene; it inhibits oncogene activation by preventing WNT signaling. This study investigated the epigenetic regulation of WIF1 gene in bladder cancer. We observed a positive relationship between WIF1 mRNA expression and survival probability of bladder cancer patients. The WIF1 gene expression could be enhanced by DNA demethylation drug 5-aza-2′-deoxycytidine (5-aza-dC) and histone deacetylase inhibitor trichostatin A (TSA), suggesting that epigenetic modifications could regulate WIF1 gene expression. Overexpression of WIF1 inhibited cell proliferation and migration in 5637 cells, confirming the tumor suppressor role of WIF1. 5-Aza-dC dose dependently increased WIF1 gene expression while reducing DNA methylation level, suggesting that reversing WIF1 DNA methylation could activate its gene expression. We collected the cancer tissues and urine pellets of bladder cancer patients and only urine pellets from non-bladder cancer volunteers for DNA methylation analysis, but the methylation level of WIF1 gene −184 to +29 did not differ between patients and controls. We also analyzed glutathione S-transferase Mu 5 (GSTM5) gene methylation level because GSTM5 DNA hypermethylation was suggested to be a tumor biomarker in our previous study. It confirmed a higher GSTM5 DNA methylation in bladder cancer patients than in controls. In summary, this study suggests that the 5-aza-dC activated WIF1 gene which showed an anti-cancer effect, while WIF1 promoter −184 to +29 did not provide a suitable methylation assay region in clinical samples. In contrast, GSTM5 promoter −258 to −89 is a useful region for DNA methylation assay because it shows a higher methylation level in bladder cancer patients.

RationaleCilostazol, an anti-platelet phosphodiesterase-3 inhibitor used for the treatment of intermittent claudication, is known for its pleiotropic effects on platelets, endothelial cells and smooth muscle cells. However, how cilostazol impacts the endocrine system and the injury-induced inflammatory processes remains unclear.MethodsWe used the zebrafish, a simple transparent model that demonstrates rapid development and a strong regenerative ability, to test whether cilostazol influences heart rate, steroidogenesis, and the temporal and dosage effects of cilostazol on innate immune cells during tissue damage and repair.ResultsWhile dosages of cilostazol from 10 to 100 μM did not induce any noticeable morphological abnormality in the embryonic and larval zebrafish, the heart rate was increased as measured by ImageJ TSA method. Moreover, adrenal/interrenal steroidogenesis in larval zebrafish, analyzed by whole-mount 3β-Hsd enzymatic activity and cortisol ELISA assays, was significantly enhanced. During embryonic fin amputation and regeneration, cilostazol treatments led to a subtle yet significant effect on reducing the aggregation of Mpx-expressing neutrophil at the lesion site, but did not affect the immediate injury-induced recruitment and retention of Mpeg1-expressing macrophages.ConclusionsOur results indicate that cilostazol has a significant effect on the heart rate and the growth as well as endocrine function of steroidogenic tissue; with a limited effect on the migration of innate immune cells during tissue damage and repair.

Waldenström Macroglobulinemia (WM) is a rare chronic lymphoproliferative disease, accounting for less than 2% of hematological malignancies. It is characterized by plasma cytoid lymphocyte infiltration in bone marrow and abnormal increase of monoclonal IgM in peripheral blood. Only 5%–10% of cases of WM secrete monoclonal IgG and IgA components or do not secrete monoclonal long immunoglobulin. This case is the first to report of serum protein recombination from lgM and Igkappa band mutation to abnormal lgG and Igkappa band after 6 years of treatment in a male patient with WM.

The haskap (Lonicera caerulea L., Caprifoliaceae) berry has been widely used in traditional medicine in Kuril Islands, Russia, Japan, and China. Cyanidin-3-O-glucoside (C3G) is the most abundant anthocyanin in haskap berries, and C3G induces antiproliferative pharmacological activity in various cancer cells. However, no study has investigated its anti-lung large-cell carcinoma (LCC) pharmacological role. Therefore, this study determined whether C3G alone or C3G combined with 5-fluorouracil (5-FU) inhibits human lung LCC. We determined the tumor growth, apoptosis, inflammation, and metastasis in the H661 lung LCC lines xenografted into BALB/c nude mice. The mice were administered saline (control), 5-FU, C3G, or both C3G and 5-FU. Relative to the control mice, those treated with C3G alone or both C3G and 5-FU exhibited impaired tumor growth; increased tumor apoptosis; decreased inflammatory cytokine levels (e.g., IL-1β, TNF-α, C-reactive protein, and IL-6); decreased inflammation-related factors, including cyclooxygenase-2 protein and nuclear factor-κB (NF-κB) mRNA; increased inhibition of NF-κB kinase α mRNA; and downregulated metastasis-related factors, such as transforming growth factor-β, CD44, epidermal growth factor receptor, and vascular endothelial growth factor. In addition, C3G alone or combined with 5-FU affected the expression of the tumor microenvironment-related factors Ki67, CD45, PDL1, and CD73. Compared with the mice treated with 5-FU or C3G alone, those treated with both C3G and 5-FU exhibited significantly impaired tumor growth, decreased tumor sizes, and increased tumor inhibition. This in vivo study demonstrated that C3G alone or combined with 5-FU may impair the growth of lung LCC and inhibit tumorigenesis. The findings indicate that C3G alone or C3G combined with 5-FU may be beneficial for treating human lung LCC.

Glutathione S-transferases (GSTs) belong to one class of phase 2 detoxification enzymes which are important in metabolism and/or detoxification of various electrophilic endogenous metabolites and xenobiotics. From the available database, we found that GSTM2 gene expression is lower in high stages of bladder urothelial carcinoma than in stage 1 and normal bladder tissue. GSTM2 overexpression retards invasion, migration and tumor sphere formation of bladder cancer cells. Analysis of GSTM2 promoter activity shows that one SP1 site located at − 48 to − 40 bp is important for GSTM2 gene expression in BFTC 905 cells. An SP1 inhibitor, mithramycin A, inhibits GSTM2 promoter activity and protein expression. SP1 overexpression also increases GSTM2 expression in BFTC 905 and 5637 cells. Eight potential phytochemicals were analyzed for GSTM2 promoter activation, and results indicated that baicalein, berberrubine, chalcone, curcumin, resveratrol, and wogonin can increase promoter activity. In endogenous GSTM2 expression, berberrubine and resveratrol activated GSTM2 mRNA and protein expression the most. A DNA methylation inhibitor, 5-aza-deoxycytidine, can decrease GSTM2 gene methylation level and then increase its gene expression; 50 μM berberrubine decreased the GSTM2 gene methylation level, providing a mechanism for activating GSTM2 gene expression. Berberrubine and resveratrol also increased SP1 protein expression as one of the mechanisms for GSTM2 gene expression. In summary, berberrubine and resveratrol activates GSTM2 expression which inhibits cell proliferation, migration, and invasion of bladder cancer cells. The GSTM2 expression mechanism is partially via SP1 activation, and the effect of berberrubine is also partly via DNA CpG demethylation.

Abstract Background Working in an underground tunnel environment is unavoidable in professions such as miners and tunnel workers, and there is a concern about the health of these workers. Few studies have addressed alterations in the intestinal microbiome of workers within that environment. Results Fecal samples were collected from the workers before they entered the tunnel (baseline status, BS) and after they left the tunnel (exposed status, ES), respectively (a time period of 3 weeks between them). We analyzed 16S rRNA sequencing to show the changes in microbial composition and self-evaluation of mental health questionnaire was also performed. The results showed that Shannon and Simpson indices decreased significantly from BS to ES. A higher abundance was found in the phylum Actinobacteria, classes Actinobacteria and Deltaproteobacteria, orders Bifidobacteriales, Coriobacteriales, and Desulfovibrionales, families Bifidobacteriaceae, Peptostreptococcaceae, Coriobacteriaceae, Clostridiaceae_1, Desulfovibrionaceae, Pseudomonadaceae, and Microbacteriaceae, and genera Bifidobacterium, Romboutsia, Clostridium sensu stricto, and Leucobacter in ES, while BS showed greater levels of genera Faecalibacterium and Roseburia. The self-evaluation showed that at least one-half of the tunnel workers experienced one or more symptoms of mental distress (inattention, sleeplessness, loss of appetite, headache or dizziness, irritability) after working in the underground tunnel environment. Conclusions Collectively, the underground tunnel environment led to alterations in the intestinal microbiome, which might be relevant to symptoms of mental distress in underground-tunnel workers.

The distortion-product otoacoustic emission (DPOAE) is a backward propagating wave generated inside the cochlea during the wave amplification process. The DPOAE signal can be detected rapidly under relatively noisy conditions. In recent years, the earphone industry demonstrated interest in adopting DPOAE as an add-on feature to make their product “intelligent” of inner-ear status. However, a technical challenge remains to be tackled—the loudspeaker in an earphone generates its own cubic distortion at the same frequency as DPOAE. Unfortunately, the intensity of loudspeaker distortion is typically comparable to that of the DPOAE, if not higher. In this research, we propose two strategies, namely compensation and cancellation, to enable DPOAE measurement with a single loudspeaker. The compensation strategy exploits the part of the growth function of the loudspeaker distortion which is almost linear, and thus suppresses the distortion it generates while retaining a larger portion of DPOAE in the residual signal. The cancellation strategy utilizes a one-dimensional Volterra filter to remove the cubic distortion from the loudspeaker. Testing on normal-hearing ears shows that the compensation strategy improved the DPOAE-to-interference ratio by approximately 7 dB, resulting in a cross-correlation of 0.62 between the residual DPOAE level and the true DPOAE level. Meanwhile, the cancellation strategy directly recovered both the magnitude and the phase of DPOAE, reducing the magnitude estimation error from 15.5 dB to 3.9 dB in the mean-square sense. These pilot results suggest that the cancellation strategy may be suitable for further testing with more subjects.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the world. It has been the most prevalent malignancy in Taiwan for consecutive thirteen years. Despite the diversity of its etiologic and pathophysiologic factors, a biological process named as epithelial-mesenchymal transition (EMT) is indispensable in the progression of epithelial cancer. Our aim is to investigate the correlation between the expression of 8 EMT-related proteins (E-cadherin, β-catenin, claudin-1, CD44, N-cadherin, fibronectin, vimentin, S100A4) and the clinicopathologic features of CRC in Taiwan, along with the DNA CpG epigenetic status of CD44 gene. In immunohistochemical assessment, decreased expression of E-cadherin is statistically associated with the progression of cancer stage, while decreased expression of claudin-1 as well as increased β-catenin nuclear translocation and N-cadherin expression is statistically associated with the progression of histopathologic grade. E-cadherin, nuclear β-catenin and claudin-1 are also associated with other important prognostic factors, including nodal metastasis, tumor deposits, and elevated serum CA 19-9 levels. In addition, the left-sided colon and rectal cancers show increased nuclear translocation of β-catenin compared to the right-sided colon cancers, while the rectal cancers show increased fibronectin expression compared to the right-sided and left-sided colon cancers. Moreover, vimentin is aberrantly expressed in one case of signet-ring cell carcinoma. The DNA methylation levels of CD44 gene promoter between the tumoral and non-tumorous tissues by NGS comparison showed statistical difference on six CpG sites. However, such difference may not be sufficient because these DNA methylation proportions are too low to inactivate CD44 gene. Our results demonstrate the expression of E-cadherin, claudin-1, and nuclear β-catenin is closely related to the clinicopathologic prognostic determinants of CRC in Taiwan. The DNA methylation level of CD44 gene and its protein expression, however, show no correlation with the clinicopathologic features in CRC.

In this study, ZnO, AlZnO, Al2O3, and Al2O3-doped ZnO-sensing membranes were fabricated in electrolyte–insulator–semiconductor (EIS) structures. Multiple material analyses indicate that annealing at an appropriate temperature of 500 °C could enhance crystallizations, passivate defects, and facilitate grainizations. Owing to their material properties, both the pH-sensing capability and overall reliability were optimized for these four types of membranes. The results also revealed that higher Al amounts increased the surface roughness values and enhanced larger crystals and grains. Higher Al compositions resulted in higher sensitivity, linearity, and stability in the membrane.

Background/Aims: 2-O-methylmagnolol (MM1), a derivative of magnolol bearing one methoxy moiety, has been shown to display improved anti-tumor activity against skin cancers. In this study, we examined the anti-tumor effects of magnolol and MM1 on oral squamous cell carcinoma (OSCC). Methods: Trypane blue staining and clonogenic assays were performed to determine the cytotoxic effects of magnolol and MM1 in OSCC cells. Migration and matrigel invasion assays were carried out to examine the metastasis effects of magnolol and MM1 in OSCC cells. IL6-stimulation, Western blot, and immunohistochemistry were used to investigate the IL-6/STAT3 signaling and apoptosis. A bioluminescent mouse model of orthotopically implanted SAS cells was used to determine the anti-tumor activity of MM1 in vivo. Results: MM1 displays greater activity than magnolol on affecting the cytotoxicity, migration, and invasion of OSCC cells cultured in vitro. The improved anti-tumor activity of MM1 was shown to associate with its greater activity to inhibit STAT3 signaling and to induce apoptosis in the OSCC. In addition, we presented evidence that MM1 is effective in inhibiting the growth of orthotopic implanted OSCC cells in vivo. Conclusion: Our data indicate that MM1 displays greater anti-tumor activity than magnolol in OSCC and is an attractive agent to be further explored for its potential clinical application.

Our previous study demonstrated that the glutathione S-transferase Mu 5 (GSTM5) gene is highly CpG-methylated in bladder cancer cells and that demethylation by 5-aza-dC activates GSTM5 gene expression. The aim of the present study was to investigate the role of GSTM5 in bladder cancer. The levels of GSTM5 gene expression and DNA methylation were analyzed in patients with bladder cancer, and functional studies of GSTM5 were conducted using GSTM5 overexpression in cultured bladder cancer cells. Clinical analysis revealed that the GSTM5 mRNA expression was lower in bladder cancer tissues than in normal tissues and that the level of GSTM5 DNA methylation was higher in bladder cancer tissues than in normal urine pellets. Overexpression of GSTM5 decreased cell proliferation, migration and colony formation capacity. Glutathione (GSH) assay results indicated that cellular GSH concentration was decreased by GSTM5 expression and that GSH supplementation reversed the decrease in proliferation and migration of cells overexpressing GSTM5. By contrast, a GSH synthesis inhibitor significantly decreased 5637 cell GSH levels, survival and migration. Furthermore, GSTM5 overexpression inhibited the adhesion of cells to the extracellular matrix protein fibronectin. To elucidate the effect of GSTM5 on anticancer drugs used to treat bladder cancer, cellular viability was compared between cells with or without GSTM5 overexpression. GSTM5-overexpressed cells showed no significant change in the cytotoxicity of cisplatin or mitomycin C in 5637, RT4 and BFTC 905 cells. Though a degree of resistance to doxorubicin was noted in 5637 cells overexpressing GSTM5, no such resistance was observed in RT4 and BFTC 905 cells. In summary, GSTM5 plays a tumor suppressor role in bladder cancer cells without significantly affecting chemoresistance to cisplatin and mitomycin C, and the cellular GSH levels highlight a key mechanism underlying the cancer inhibition effect of GSTM5. These findings suggest that low gene expression and high DNA methylation levels of GSTM5 may act as tumor markers for bladder cancer.

The objective of this study was to synthesize the 9-/13-position substituted berberine derivatives and evaluate their cytotoxic and photocytotoxic effects against three human cancer cell lines. Among all the synthesized compounds, 9-O-dodecyl- (5e), 13-dodecyl- (6e), and 13-O-dodecyl-berberine (7e) exhibited stronger growth inhibition against three human cancer cell lines, (HepG2, HT-29 and BFTC905), in comparison with structurally related berberine (1). These three compounds also showed the photocytotoxicity in human cancer cells in a concentration-dependent and light dose-dependent manner. Through flow cytometry analysis, we found out a lipophilic group at the 9-/13-position of berberine may have facilitated its penetration into test cells and hence enhanced its photocytotoxicity on the human liver cancer cell HepG2. Further, in cell cycle analysis, 5e, 6e, and 7e induced HepG2 cells to arrest at the S phase and caused apoptosis upon irradiation. In addition, photodynamic treatment of berberine derivatives 5e, 6e, and 7e again showed a significant photocytotoxic effects on HepG2 cells, induced remarkable cell apoptosis, greatly increased intracellular ROS level, and the loss of mitochondrial membrane potential. These results over and again confirmed that berberine derivatives 5e, 6e, and 7e greatly enhanced photocytotoxicity. Taken together, the test data led us to conclude that berberine derivatives with a dodecyl group at the 9-/13-position could be great candidates for the anti-liver cancer medicines developments.

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome.

Clostridium difficile is considered to be one of the major cause of infectious diarrhea in healthcare systems worldwide. Symptoms of C. difficile infection are caused largely by the production of two cytotoxins: toxin A (TcdA) and toxin B (TcdB). Vaccine development is considered desirable as it would decrease the mounting medical costs and mortality associated with C. difficile infections. Biodegradable nanoparticles composed of poly-γ-glutamic acid (γ-PGA) and chitosan have proven to be a safe and effective antigen delivery system for many viral vaccines. However, few studies have used this efficient antigen carrier for bacterial vaccine development. In this study, we eliminated the toxin activity domain of toxin B by constructing a recombinant protein rTcdB consists of residues 1852-2363 of TcdB receptor binding domain. The rTcdB was encapsulated in nanoparticles composed of γ-PGA and chitosan. Three rounds of intraperitoneal vaccination led to high anti-TcdB antibody responses and afforded mice full protection mice from lethal dose of C. difficile spore challenge. Protection was associated with high levels of toxin-neutralizing antibodies, and the rTcdB-encapsulated NPs elicited a longer-lasting antibody titers than antigen with the conventional adjuvant, aluminum hydroxide. Significant reductions in the level of proinflammatory cytokines and chemokines were observed in vaccinated mouse. These results suggested that polymeric nanocomplex-based vaccine design can be useful in developing vaccine against C. difficile infections.

The bladder is an important organ for the storage of excreted water and metabolites. If metabolites with carcinogenic characteristics are present in urine, the urothelial lining of the bladder could be damaged and genetically altered. In this study, we analyzed the interaction of arsenic and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) on mouse bladder carcinogenesis. Our previous study found that arsenic affects BBN-altered urothelial enzymatic activity, protein expression, DNA oxidation and global DNA CpG methylation levels. In this study, two mouse models were used. First, after administering a co-treatment of BBN and arsenic for 20 weeks, BBN alone led to a urothelial carcinoma formation of 20%, and arsenic promoted a BBN-induced urothelial carcinoma formation of 10%. The protein expression of GSTM1, GSTO1, NQO1, and p21 did not change by arsenic along with the BBN co-treatment, but the Sp1 expression increased. In the second mouse model, BBN was a pretreatment promoter; arsenic dose-dependently deteriorated BBN-promoted dysplasia by 10% and 40% at 10 ppm and 100 ppm, respectively. Conversely, BBN pretreatment also accelerated arsenic-induced dysplasia by 30%. The urothelial carcinogenic effect reversed after ceasing BBN for a period of 20 weeks. In summary, three conclusions were drawn from this study. The first is the mutual promotion of arsenic and BBN in bladder carcinogenesis. Second, arsenic dosages without bladder carcinogenicity (10 ppm) or with slight carcinogenicity (100 ppm) promote BBN-induced mice bladder cancer progression. Finally, the dysplastic urothelium had reverted to near-normal morphology after ceasing BBN intake for 20 weeks, providing a good suggestion for people who want to quit smoking.

Bladder cancer is highly recurrent after therapy, which has an enormous impact on the health and financial condition of the patient. It is worth developing diagnostic tools for bladder cancer. In our previous study, we found that the bladder carcinogen BBN increased urothelial global DNA CpG methylation and decreased GSTM1 protein expression in mice. Here, the correlation of BBN-decreased GSTM1 and GSTM gene CpG methylation status was analyzed in mice bladders. BBN treatment decreased the protein and mRNA expression of GSTM1, and the CpG methylation ratio of GSTM1 gene promoter was slightly increased in mice bladders. Unlike mouse GSTM1, the human GSTM1 gene tends to be deleted in bladder cancers. Among 7 human bladder cancer cell lines, GSTM1 gene is really null in 6 cell lines except one, T24 cells. The CpG methylation level of GSTM1 was 9.9% and 5-aza-dC did not significantly increase GSTM1 protein and mRNA expression in T24 cells; however, the GSTM5 gene was CpG hypermethylated (65.4%) and 5-aza-dC also did not affect the methylation ratio and mRNA expression. However, in other cell lines without GSTM1, 5-aza-dC increased GSTM5 expression and decreased its CpG DNA methylation ratio from 84.6% to 61.5% in 5637, and from 97.4% to 75% in J82 cells. In summary, two biomarkers of bladder tumor were provided. One is the GSTM1 gene which is down-regulated in mice bladder carcinogenesis and is usually deleted in human urothelial carcinoma, while the other is the GSTM5 gene, which is inactivated by DNA CpG methylation.

BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms. CONCLUSIONS AND SIGNIFICANCE: These results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.

BACKGROUND: The early morphogenetic steps of zebrafish interrenal tissue, the teleostean counterpart of the mammalian adrenal gland, are modulated by the peri-interrenal angioblasts and blood vessels. While an organized distribution of intra-adrenal vessels and extracellular matrix is essential for the fetal adrenal cortex remodeling, whether and how an intra-interrenal buildup of vasculature and extracellular matrix forms and functions during interrenal organogenesis in teleosts remains unclear. METHODOLOGY AND PRINCIPAL FINDINGS: We characterized the process of interrenal gland vascularization by identifying the interrenal vessel (IRV); which develops from the axial artery through angiogenesis and is associated with highly enriched Fibronectin (Fn) accumulation at its microenvironment. The loss of Fn1 by either antisense morpholino (MO) knockdown or genetic mutation inhibited endothelial invasion and migration of the steroidogenic tissue. The accumulation of peri-IRV Fn requires Integrin α5 (Itga5), with its knockdown leading to interrenal and IRV morphologies phenocopying those in the fn1 morphant and mutant. fn1b, another known fn gene in zebrafish, is however not involved in the IRV formation. The distribution pattern of peri-IRV Fn could be modulated by the blood flow, while a lack of which altered angiogenic direction of the IRV as well as its ability to integrate with the steroidogenic tissue. The administration of Fn antagonist through microangiography exerted reducing effects on both interrenal vessel angiogenesis and steroidogenic cell migration. CONCLUSIONS AND SIGNIFICANCE: This work is the first to identify the zebrafish IRV and to characterize how its integration into the developing interrenal gland requires the Fn-enriched microenvironment, which leads to the possibility of using the IRV formation as a platform for exploring organ-specific angiogenesis. In the context of other developmental endocrinology studies, our results indicate a highly dynamic interrenal-vessel interaction immediately before the onset of stress response in the zebrafish embryo.