Study on the Role and Mechanism of SLC3A2 in Tumor-Associated Macrophage Polarization and Bladder Cancer Cells Growth.

Background: Solute carrier family 3 member 2 (SLC3A2) is highly expressed in various types of cancers, including bladder cancer (BLCA). However, the role and mechanism of SLC3A2 in the onset and progression of BLCA are still unclear. Methods: The interfering plasmid for SLC3A2 was constructed and transfected into BLCA cells. Cell proliferation, invasion, and migration abilities were assessed to evaluate the impact of SLC3A2 silencing on BLCA cell growth. M1 and M2 macrophage polarization markers were detected to evaluate macrophage polarization. The levels of reactive oxygen species (ROS), lipid peroxidation, and Fe2+, as well as the expression of ferroptosis-related proteins, were measured to assess the occurrence of ferroptosis. Ferroptosis inhibitors were used to verify the mechanism. Results: The experimental results showed that SLC3A2 was highly expressed in BLCA cell lines. The proliferation, invasion, and migration of BLCA cells were reduced after interfering with SLC3A2. Interference with SLC3A2 led to increase the expression of M1 macrophage markers and decreased the expression of M2 macrophage markers in M0 macrophages co-cultured with tumor cells. Additionally, interference with SLC3A2 led to increased levels of ROS, lipid peroxidation, and Fe2+, downregulated the expression of solute carrier family 7 member11 (SLC7A11) and glutathione peroxidase 4 (GPX4), while upregulated the expression of acyl-coA synthetase long chain family member 4 (ACSL4) and transferrin receptor 1 (TFR1) in BLCA cells. However, the impact of SLC3A2 interference on cell proliferation and macrophage polarization was impeded by ferroptosis inhibitors. Conclusion: Interference with SLC3A2 inhibited the growth of BLCA cells and the polarization of tumor-associated macrophages by promoting ferroptosis in BLCA cells.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1.

BACKGROUND: Macrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. METHODS: We used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE-/-- mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. RESULTS: We determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. CONCLUSION: Macrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a.

Accumulating evidence has shown that long noncoding RNAs (lncRNAs) are involved in several biological processes, including immune responses. However, the role of lncRNAs in antiviral innate immune responses remains largely elusive. Here, we identify an uncharacterized human lncRNA AVAN from influenza A virus (IAV) infected patients, that is significantly upregulated following RNA virus infection. During IAV infection, AVAN play an indispensable role in antiviral immune responses. In vivo, we enforced the expression of AVAN in transgenic mice or adeno-associated virus encoding AVAN delivery system and found that AVAN significantly alleviated IAV virulence and virus replication. Mechanistically, nuclear AVAN positively regulates the transcription of forkhead box O3A (FOXO3a) by associating with its promoter and inducing chromatin remodeling to promote neutrophil chemotaxis. Meanwhile, cytoplasmic AVAN binds directly to the E3 ligase TRIM25 and enhances TRIM25-mediated K63-linked ubiquitination of RIG-I, thereby promoting TRIM25- and RIG-I-mediated antiviral innate immune responses, including the induction of type I interferon and ISGs. Moreover, AVAN binds to the B Box/CCD domain of TRIM25 and 1-200nt of AVAN were the functional moieties. Collectively, our findings highlight the potential clinical implications of human lncRNA AVAN as a key positive regulator of the antiviral innate immune response and a promising target for developing broad antiviral therapeutics.

Effects of Acute Sacral Neuromodulation at Different Pulse Widths on Bladder Overactivity in Pigs.

PURPOSE: Sacral nerve stimulation has been used to treat overactive bladder. This study evaluated the effects of stimulation using different pulse widths on the inhibition of bladder overactivity by sacral nerve stimulation (SNM) in pigs. METHODS: Implant-driven stimulators were used to stimulate the S3 spinal nerve in 7 pigs. Cystometry was performed by infusing normal saline (NS) or acetic acid (AA). SNM at pulse widths of 64 µsec to 624 µsec was conducted at the intensity threshold at which observable perianal and/or tail movement was induced. Multiple cystometrograms were performed to determine the effects of different pulse widths on the micturition reflex. RESULTS: AA-induced bladder overactivity reduced the bladder capacity to 46.9%±7.1% of the NS control level (P<0.05). During AA infusion, SNM at 64 µsec, 204 µsec, and 624 µsec increased the bladder capacity to 126.1%±6.9%, 129.5%±7.3%, and 140.1%±7.6% of the AA control level (P<0.05). No significant differences were found among the results obtained using pulse widths of 64 µsec, 204 µsec, and 624 µsec (P>0.05). The actual intensity threshold varied from 0.7 to 8 V. The mean intensity threshold (T visual) for pulse widths of 64 µs, 204 µs, and 624 µs were 5.64±0.76 V, 3.11±0.48 V, and 2.52±0.49 V. T visual for pulse widths of 64 µsec was larger than the other two T visual for pulse widths of 204 µsec and 624 µsec (P<0.05). No significant differences were found among the T visual for pulse widths of 204 µsec and 624 µsec (P>0.05). CONCLUSION: This study indicated that different pulse widths could play a role in inhibiting bladder overactivity. It is not yet certain which pulse widths increased bladder capacity compared with AA levels, to minimize energy consumption and maintain patient comfort during stimulation, 204 µsec may be an appropriate pulse width for SNM.

Evaluation of sacral neuromodulation system with new six-contact points electrode in pigs.

AIMS: We investigated the effects of sacral neuromodulation using the new six-contact electrode vs the four-contact electrode in pigs. METHODS: Randomly, a six-contact electrode was implanted in eight pigs in one side of the third sacral (S3) foramen, and a four-contact electrode was implanted in the other side using the same method. Using an external neurostimulator, the number of contact points (sensitive voltage ≤ 2 V) of both electrodes (SacralStim and InterStim systems) was calculated. Cystometry was performed by infusing normal saline or acetic acid. Then sacral neuromodulation with the SacralStim and InterStim systems was induced at a voltage at which we could observe perianal and/or tail movement. Multiple cystometrograms were performed to determine the effects of the two systems on the micturition reflex. RESULTS: The mean number of sensitive points of six-contact electrodes of the SacralStim system (2.63 ± 0.32) was higher than that of the quadripolar-lead electrodes of the InterStim system (1.38 ± 0.18), and the difference was statistically significant (P < 0.05). Acetic acid-induced bladder overactivity significantly reduced bladder capacity to 54.89% ± 4.7% of the normal saline control level. During acetic acid infusion, sacral neuromodulation with the SacralStim system suppressed bladder overactivity and significantly increased bladder capacity to 70.41% ± 5.4% of the normal saline control level, compared with the acetic acid level ( P < 0.05). Moreover, sacral neuromodulation with the InterStim system also significantly increased bladder capacity to 69.63% ± 5.3% of the normal saline control level, compared with the acetic acid level ( P < 0.05). No significant differences were found in the results obtained using the two systems ( P > 0.05). CONCLUSIONS: The six-contact electrode of the SacralStim system had more sensitive points (<2 V) than that of the quadripolar-lead electrode of InterStim system. Potentially, it has more postimplantation programming options and battery savings manifested by lower voltage will increase the longevity of the stimulator. Further studies of sacral neuromodulation with six-contact electrodes in clinical practice are needed.

Effects of bladder shape on accuracy of measurement of bladder volume using portable ultrasound scanner and development of correction method.

AIMS: To compare the accuracy of using a bladder scanner to measure bladder volume through intermittent catheterization (IC) in patients and to introduce the Bladder Deformation Index (BDI) to develop a correction method. METHODS: Bladder volume was assessed by a nurse with the scanner. A second nurse catheterized the patient's bladder. A third nurse measured the urine volume in a 500-mL or 1000-mL graduated cylinder. RESULTS: Sixty one patients were included and 590 pairs of data were obtained. The mean bladder volume measured using a scanner and IC was (332.3 ± 156.1) mL and (339.1 ± 158.8) mL. The mean absolute difference was 30.8 mL. The correlation coefficient was 0.929. Patients were classified into 2 groups depending on whether they had undergone augmentation cystoplasty. The mean absolute difference was 109.2 and 20.4 mL. The correlation coefficient was 0.712 and 0.981. According to the BDI, bladders can be classified into 3 groups. The mean absolute difference was 21.9, 60.4, and 109.4 mL. The correlation coefficient was 0.970, 0.839, and 0.783. The linear regression equations of Grade I and Grade II were Y = 1.11X + 3.1 and Y = 0.76X + 161.5. CONCLUSIONS: The results showed that bladder shape plays a critical role in accuracy which is inversely associated with BDI. This degree of accuracy is sufficient; especially measurement adjusted using the linear regression equation in patients with high BDI. However, although the preliminary results of the study are promising, a large-scale prospective study should be needed to address the validation of the data in the future.

Bivalent vaccine platform based on ca influenza virus vaccine elicits protective immunity against human adenoviruses.

Human adenoviruses (HAdVs) are prevalent in pediatric and adult patients with severe acute respiratory disease (ARD). To date, there have been no widely used HAdV vaccines available. In this report, we developed a cold-adapted attenuated influenza virus, termed rg HAdV-Flu ca, carrying epitopes from HAdV hexon protein in the backbone of the ca influenza vaccine neuraminidase (NA) gene using reverse genetics. Rg HAdV-Flu ca virus exhibited a cold-adapted (ca) phenotype, and its morphological characteristics were observed using electron microscopy. Moreover, BALB/c mice were immunized intranasally (i.n.) with 105, 106 or 107 TCID50 rg HAdV-Flu ca. Results showed a specific, robust antibody response against influenza and HAdV in a dose-dependent manner. More importantly, potent humoral, mucosal and cellular immune responses protected against subsequent wild-type HAdV-3 or HAdV-7 challenges, as determined by a significant decrease in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice. These findings demonstrate that rg HAdV-Flu ca warrants attention as a potential vaccine candidate against HAdV infection.

Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus.

BACKGROUND: Egg drop syndrome (EDS), caused by the adenovirus "egg drop syndrome virus" (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV. RESULTS: For our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40-45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses. CONCLUSIONS: The direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

The roles of connective tissue growth factor in the development of anastomotic esophageal strictures.

INTRODUCTION: The aim of this study was to investigate the roles of connective tissue growth factor (CTGF) in the development of anastomotic strictures after surgical repair of the esophagus. MATERIAL AND METHODS: Tissues collected from the patients were divided into three groups based on the results of endoscopy and clinical grading. Patients without dysphagia after esophagectomy were used as the control population. The protein levels of CTGF, TGF-ß1, Smad2, and Smad4 were determined by immunohistochemistry (IHC) and western blot analyses, while the mRNA levels of the two growth factors were evaluated by real-time polymerase chain reaction. RESULTS: Compared with the control group, significantly increased (p < 0.01) levels of CTGF and TGF-ß1 protein were observed in the anastomotic stenosis (AS) group, and levels of the two proteins detected by the IHC and western blot analyses were also significantly increased with the increasing severity of stenosis (p < 0.05). The mRNA levels of CTGF and TGF-ß1 in the tissues collected from the patients with stenosis were significantly up-regulated (p < 0.05) as compared with those from the control group. In addition, the levels of Smad2 and Smad4 protein were also significantly increased (p < 0.05) with the increasing severity of stenosis, and the protein levels were positively correlated with the levels of CTGF (r = 0.59, p < 0.05) and TGF-ß1 (r = 0.63, p < 0.05). CONCLUSIONS: Inhibition of CTGF protein or mRNA expression may be a distinctive and effective therapy for the treatment of postoperative anastomotic strictures.