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.

A novel lncRNA DFRV plays a dual function in influenza A virus infection.

Long noncoding RNAs (lncRNAs) have been associated with a variety of biological activities, including immune responses. However, the function of lncRNAs in antiviral innate immune responses are not fully understood. Here, we identified a novel lncRNA, termed dual function regulating influenza virus (DFRV), elevating in a dose- and time-dependent manner during influenza A virus (IAV) infection, which was dependent on the NFκB signaling pathway. Meanwhile, DFRV was spliced into two transcripts post IAV infection, in which DFRV long suppress the viral replication while DFRV short plays the opposite role. Moreover, DFRV regulates IL-1ß and TNF-α via activating several pro-inflammatory signaling cascades, including NFκB, STAT3, PI3K, AKT, ERK1/2 and p38. Besides, DFRV short can inhibit DFRV long expression in a dose-dependent manner. Collectively, our studies reveal that DFRV may act as a potential dual-regulator to preserve innate immune homeostasis in IAV infection.

Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2.

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

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.

Brain functional connectivity during storage based on resting state functional magnetic resonance imaging with synchronous urodynamic testing in healthy volunteers.

The aim of the study was to elucidate the correlation between spatially distinct brain areas with a full bladder from the perspective of functional connectivity using resting-state functional magnetic resonance imaging (rs-fMRI) with simultaneous urodynamic testing in healthy volunteers. The brain regions with full and empty bladders were reported via rs-fMRI using a 3 T magnetic resonance system. Then, we identified brain regions that are activated during bladder filling by calculating the amplitude of low-frequency fluctuation (ALFF) values using brain imaging software (DPABI and SPM8) and empirically derived six regions of interest (ROI) from analysis of activation were used as seeds for resting-state functional connectivity (rs-FC) analysis with the rest of the brain to examine differences in the two conditions. Statistical analysis was performed with a paired t-test and statistical significance was defined as a P < 0.01. Twenty-two healthy volunteers (11 men and 11 women) 35-64 years of age were enrolled. The rs-fMRI scans of 22 healthy volunteers were analyzed. After motion correction, two subjects were excluded. Meaningful data were obtained on 20 of these subjects. Compared with an empty bladder, functional connection enhancement was noted mainly in the right inferior orbitofrontal cortex and bilateral calcarine gyrus, the left lingual gyrus, left fusiform gyrus, left superior occipital gyrus, right insula, right inferior temporal gyrus, superior parietal lobe, left insula, right lingual gyrus, right fusiform gyrus, left parahippocampal gyrus, right inferior temporal gyrus, superior parietal lobe, left calcarine gyrus, bilateral lingual gyrus, prefrontal cortex, including the middle frontal gyrus and superior frontal gyrus, the right middle temporal gyrus, bilateral posterior cingulate cortex, and right precuneus. The decrease in functional connection was mainly located in the right inferior orbitofrontal cortex, prefrontal cortex, including the superior frontal gyrus, orbitofrontal cortex, and anterior cingulate cortex, the left inferior orbitofrontal cortex, right insula, middle occipital gyrus, angular gyrus, inferior frontal gyrus, right insula, middle temporal gyrus, inferior parietal lobe, middle occipital gyrus, supplementary motor area, superior frontal gyrus, left insula, bilateral posterior cingulate cortex, bilateral precuneus, middle occipital gyrus, and right middle temporal lobe. There were significant changes in the functional connectivity of the brain between full and empty bladders in healthy volunteers, which suggests that the central neural processes involved in storage needs brain areas with integrated control. These findings are strong evidence for physicians to consider brain responses in urine storage and offer the provision of some normative data.

A Long Non-coding RNA IVRPIE Promotes Host Antiviral Immune Responses Through Regulating Interferon ß1 and ISG Expression.

Accumulating studies have shown that long non-coding RNAs (lncRNAs) modulate multiple biological processes, including immune response. However, the underlying mechanisms of lncRNAs regulating host antiviral immune response are not well elucidated. In this study, we report that analysis of the existing dataset transcriptome of blood immune cells of patients with influenza A virus (IAV) infection and after recovery (GSE108807) identified a novel lncRNA, termed as IVRPIE (Inhibiting IAV Replication by Promoting IFN and ISGs Expression), was involved in antiviral innate immunity. In vitro studies showed that IVRPIE was significantly upregulated in A549 cells after IAV infection. Gain-and-loss of function experiments displayed that enforced IVRPIE expression significantly inhibited IAV replication in A549 cells. Conversely, silencing IVRPIE promoted IAV replication. Furthermore, IVRPIE positively regulates the transcription of interferon ß1 and several critical interferon-stimulated genes (ISGs), including IRF1, IFIT1, IFIT3, Mx1, ISG15, and IFI44L, by affecting histone modification of these genes. In addition, hnRNP U was identified as an interaction partner for IVRPIE. Taken together, our findings suggested that a novel lncRNA IVRPIE is a critical regulator of host antiviral response.

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.

Basic fibroblast growth factor protects against influenza A virus-induced acute lung injury by recruiting neutrophils.

Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2-PI3K-AKT-NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection.

miR-194 Inhibits Innate Antiviral Immunity by Targeting FGF2 in Influenza H1N1 Virus Infection.

Fibroblast growth factor 2 (FGF2 or basic FGF) regulates a wide range of cell biological functions including proliferation, angiogenesis, migration, differentiation, and injury repair. However, the roles of FGF2 and the underlying mechanisms of action in influenza A virus (IAV)-induced lung injury remain largely unexplored. In this study, we report that microRNA-194-5p (miR-194) expression is significantly decreased in A549 alveolar epithelial cells (AECs) following infection with IAV/Beijing/501/2009 (BJ501). We found that miR-194 can directly target FGF2, a novel antiviral regulator, to suppress FGF2 expression at the mRNA and protein levels. Overexpression of miR-194 facilitated IAV replication by negatively regulating type I interferon (IFN) production, whereas reintroduction of FGF2 abrogated the miR-194-induced effects on IAV replication. Conversely, inhibition of miR-194 alleviated IAV-induced lung injury by promoting type I IFN antiviral activities in vivo. Importantly, FGF2 activated the retinoic acid-inducible gene I signaling pathway, whereas miR-194 suppressed the phosphorylation of tank binding kinase 1 and IFN regulatory factor 3. Our findings suggest that the miR-194-FGF2 axis plays a vital role in IAV-induced lung injury, and miR-194 antagonism might be a potential therapeutic target during IAV infection.

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.

High-precision rotation angle measurement method based on monocular vision.

To accurately measure the attitude angles (pitch, roll, and yaw) of a rigid object that rotates in a space, we propose a high-precision rotation angle measurement method based on monocular vision. This method combines camera self-calibration, multiview geometry, and 3D measurement. This monocular vision measuring system consists of an area scan CCD, a prime lens, and a spots array target, which are fixed on the measured object. We can calculate the rotation angle according to the rebuilt rotating spots array target by using this monocular vision measuring system. The measurement precision of rotation angle can reach 1 arc sec in this paper's experiments. This method has high measurement precision and good stability. Therefore we can widely use this method in machinery manufacturing, engineering measurement, aerospace, and the military.