Urolithin A exerts a protective effect on lipopolysaccharide-induced acute lung injury by regulating HMGB1-mediated MAPK and NF-κB signaling pathways.

Acute lung injury (ALI) is a severe inflammatory disorder that has a high morbidity and mortality rate. Urolithin A (UA) is reported to have anti-inflammatory and anti-oxidative effects in ALI. However, its molecular mechanisms in ALI remain to be explored. Mice and BEAS-2B cells were administrated with lipopolysaccharide (LPS) to mimic the ALI model in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to detect the pathological injury of lung tissues. The levels of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and culture supernatant and the levels of oxidative stress markers in lung tissues were measured using ELISA. DCFH-DA probe was used to assess the reactive oxygen species (ROS) level. TUNEL staining and flow cytometry were performed to determine cell apoptosis. The key targets and pathways were confirmed by immunohistochemistry (IHC) and western blot. UA suppressed the pathologic damage, wet/dry weight ratio, and total protein and inflammatory cells in BALF. UA decreased neutrophil infiltration and proinflammatory cytokines production. UA reduced the level of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in pulmonary tissues. UA also inhibited cell apoptosis in lung tissues by decreasing Bax expression and increasing Bcl-2 expression. In addition, UA suppressed LPS-induced inflammatory factor production, ROS level, and cell apoptosis in BEAS-2B. Importantly, UA decreased the expression of HMGB1 in LPS-treated mice and BEAS-2B cells. HMGB1 overexpression greatly abrogated the inhibition of UA on inflammation, ROS, and cell apoptosis in LPS-administrated BEAS-2B. Furthermore, UA treatment suppressed the phosphorylated levels of p38, JNK, ERK, and p65 in LPS-administrated mice and BEAS-2B cells. UA alleviated lung inflammation, oxidative stress, and apoptosis in ALI by targeting HMGB1 to inactivate the MAPK/NF-κB signaling, suggesting the potential of UA to treat ALI.

Proteomic-based stratification of intermediate-risk prostate cancer patients.

Gleason grading is an important prognostic indicator for prostate adenocarcinoma and is crucial for patient treatment decisions. However, intermediate-risk patients diagnosed in the Gleason grade group (GG) 2 and GG3 can harbour either aggressive or non-aggressive disease, resulting in under- or overtreatment of a significant number of patients. Here, we performed proteomic, differential expression, machine learning, and survival analyses for 1,348 matched tumour and benign sample runs from 278 patients. Three proteins (F5, TMEM126B, and EARS2) were identified as candidate biomarkers in patients with biochemical recurrence. Multivariate Cox regression yielded 18 proteins, from which a risk score was constructed to dichotomize prostate cancer patients into low- and high-risk groups. This 18-protein signature is prognostic for the risk of biochemical recurrence and completely independent of the intermediate GG. Our results suggest that markers generated by computational proteomic profiling have the potential for clinical applications including integration into prostate cancer management.

Effect of ocean warming on pigment and photosynthetic carbon fixation of plankton assemblage in Pingtan Island of Southeast China.

Temperature plays an important role in affecting the physiological traits of marine plankton. In this study, we conducted an outdoor incubation experiment to investigate the effects of elevated temperature on Chl a, photosynthetic carbon fixation and the composition of plankton communities in the surface seawater around Pingtan Island, the northwest Taiwan Strait in Autumn 2022. After 3-4 days of incubation, elevated temperature (1-4 °C higher than ambient temperature) led to a decrease in Chl a concentration across all three stations, did not result in significant increases in the particulate organic carbon (POC) and nitrogen (PON) concentrations in seawater with high nitrate concentrations, whereas increased POC and PON concentrations in nitrate-limited seawater. These findings suggest that the effect of temperature on the POC and PON contents of plankton is affected by the availability of nitrate. Diatoms were the dominant phytoplankton group in all three stations. Our results indicate that ocean warming has a potential to increase the POC contents of marine plankton per volume of seawater, which may increase the ability of phytoplankton to absorb atmospheric CO2 and to alleviate global warming.

Multi-omics blood atlas reveals unique features of immune and platelet responses to SARS-CoV-2 Omicron breakthrough infection.

Although host responses to the ancestral SARS-CoV-2 strain are well described, those to the new Omicron variants are less resolved. We profiled the clinical phenomes, transcriptomes, proteomes, metabolomes, and immune repertoires of >1,000 blood cell or plasma specimens from SARS-CoV-2 Omicron patients. Using in-depth integrated multi-omics, we dissected the host response dynamics during multiple disease phases to reveal the molecular and cellular landscapes in the blood. Specifically, we detected enhanced interferon-mediated antiviral signatures of platelets in Omicron-infected patients, and platelets preferentially formed widespread aggregates with leukocytes to modulate immune cell functions. In addition, patients who were re-tested positive for viral RNA showed marked reductions in B cell receptor clones, antibody generation, and neutralizing capacity against Omicron. Finally, we developed a machine learning model that accurately predicted the probability of re-positivity in Omicron patients. Our study may inspire a paradigm shift in studying systemic diseases and emerging public health concerns.

Longitudinal proteomic investigation of COVID-19 vaccination.

Although the development of COVID-19 vaccines has been a remarkable success, the heterogeneous individual antibody generation and decline over time are unknown and still hard to predict. In this study, blood samples were collected from 163 participants who next received two doses of an inactivated COVID-19 vaccine (CoronaVac®) at a 28-day interval. Using TMT-based proteomics, we identified 1,715 serum and 7,342 peripheral blood mononuclear cells (PBMCs) proteins. We proposed two sets of potential biomarkers (seven from serum, five from PBMCs) at baseline using machine learning, and predicted the individual seropositivity 57 days after vaccination (AUC = 0.87). Based on the four PBMC's potential biomarkers, we predicted the antibody persistence until 180 days after vaccination (AUC = 0.79). Our data highlighted characteristic hematological host responses, including altered lymphocyte migration regulation, neutrophil degranulation, and humoral immune response. This study proposed potential blood-derived protein biomarkers before vaccination for predicting heterogeneous antibody generation and decline after COVID-19 vaccination, shedding light on immunization mechanisms and individual booster shot planning.

Integrating Network Pharmacology and Experimental Validation to Elucidate the Mechanism of Yiqi Yangyin Decoction in Suppressing Non-Small-Cell Lung Cancer.

Yiqi Yangyin Decoction (YYD) is a classic traditional Chinese medicine (TCM) formulation to treat lung cancer in clinic. Nevertheless, the active ingredients, key targets, and molecular mechanisms for YYD are still poorly understood. This study is focused on elucidating the pharmacological mechanism of YYD in non-small-cell lung cancer (NSCLC) by using a combined network pharmacology approach and biological experiment validation. Online bioinformatics tools showed that 40 bioactive compounds and 229 putative targets of YYD were associated with anti-NSCLC activity. Protein-Protein Interaction (PPI) network demonstrated AKT1, SRC, JUN, TP53, and EGFR as the top five key targets for YYD against NSCLC. Through enrichment analysis, YYD was found to affect cell proliferation and apoptosis in NSCLC possibly by PI3K-AKT signaling. Molecular docking confirmed a strong binding between the main compounds (quercetin or luteolin) and EGFR. As demonstrated by CCK-8, EdU, and colony formation assays, we found a significant inhibition of YYD on cell proliferation. Moreover, YYD treatment induced cell cycle arrest by affecting p53, p21, and cyclin D1 expression. YYD administration enhanced apoptosis by changing the expression of cleaved caspase-3, Bax, and Bcl-2. Mechanistically, YYD resulted in a significant inactivation of EGFR-PI3K-AKT signaling. Furthermore, EGFR activator significantly reversed YYD-mediated proliferation inhibition and apoptosis. YYD also showed an inhibitory effect on tumor growth in mice. Together, YYD might target the EGFR-PI3K-AKT pathway to repress NSCLC progression.

Molecular mechanism of betulin palliative therapy for chronic obstructive pulmonary disease (COPD) based on P2X7 receptor target of gated ion channel.

Background: The aim of this study was to discover the molecular mechanism of betulin palliative therapy for chronic obstructive pulmonary disease (COPD) based on the P2X7 receptor target of gated ion channel. Methods: A COPD mouse model was developed. Changes in pulmonary ventilation function, pulmonary airway and vascular remodeling indicators, inflammatory cells, and inflammatory factors were determined after betulin intervention, and the pathological alterations of lung tissues were detected. An in vitro experimental model was constructed to observe the influence of betulin at varying concentrations on the proliferation of human bronchial epidermal cell line (16-HBE) cells and changes in inflammatory factors in cell supernatant. The expression levels of key proteins in 16-HBE cells transfected with overexpressed or silenced P2X7 genes were determined through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Results: After betulin intervention, pulmonary ventilation function in the 20 mg/kg betulin and 40 mg/kg betulin groups was improved. Levels of white blood cells (WBCs), neutrophils (Ns), tumor necrosis factor (TNF), TNF-ɑ, interleukin (IL)-1ß, and IL-6 in the 2 groups also decreased significantly (all P<0.05). The pathological changes in COPD mice were detected. After betulin intervention, the pathological injury of the lung was reduced, the pathological score decreased significantly, and the remodeling indicators of pulmonary airway and pulmonary vessels diminished remarkably (all P<0.05). Betulin had no effect on the proliferation of 16-HBE cells in vitro. After cigarette smoke extract (CSE) stimulation, the rate of survival for 16-HBE cells decreased significantly. After betulin treatment, the survival rate of 16-HBE cells augmented remarkably, and the levels of TNF-ɑ, IL-6, and IL-1ß in cell supernatant reduced substantially (all P<0.05). 16-HBE with overexpression and knockdown of P2X7 was constructed. After being treated with betulin, the relative expression levels of messenger RNA (mRNA) of ERK, JNK, rho-associated protein kinase (ROCK), nuclear factor-κB (NF-κB), and p38 in 16-HBE cells with P2X7 overexpression or knockdown were decreased significantly (all P<0.05), but the above indicators were largely unchanged (all P>0.05). Conclusions: Betulin relieved lung pathological injury, ameliorated lung ventilation function, and diminished the level of inflammatory factors in COPD mice, playing a therapeutic role via the P2X7 signaling pathway.

High pressure processing improves the texture quality of fermented minced pepper by maintaining pectin characteristics during storage.

Texture quality affects the sensory and market acceptance of fermentation minced pepper (FMP), but it will deteriorate during storage. Thus, high pressure processing (HPP) and thermal pasteurization (TP) were used to improve the texture quality of FMP during storage. The results showed that variations in texture quality and pectin characteristics under HPP and TP treatments were similar during storage. The hardness, cell wall material (CWM) and sodium carbonate-soluble pectin (SSP) content, water-soluble pectin (WSP) molecular weight (Mw ) decreased, while WSP content and sodium chelate-soluble pectin (CSP) Mw increased after storage. HPP-treated FMP showed higher hardness (66.64-85.95 N) than that in TP-treated one (57.23-62.72 N) during storage. Rhamnose (Rha), arabinose, mannose, and glucose were the crucial compositions in three pectins, and their total molar ratios, respectively, reached 89.19% and 87.97% after HPP and TP treatment. However, the molar ratio of most monosaccharide in three pectins decreased after storage. Atomic force microscope images indicated the short chains and branch structures increased but aggregates decreased in most pectin components during storage. Pearson correlation analysis demonstrated FMP hardness was extremely (p < 0.01) positively correlated with CWM and SSP content, and extremely (p < 0.01) negatively correlated with WSP content. Compared to TP treatment, HPP presented higher hardness, SSP content and Mw , Rha content, CSP Mw , and lower WSP content during storage. Hence, HPP was an effective method to improve the texture quality of FMP by maintaining pectin characteristics during storage. PRACTICAL APPLICATION: Softening is one of the main factors affecting market value and consumer preferences for FMP, and it is closely related to the modification and depolymerization of pectin. Changes of texture quality and pectin properties in HPP- and TP-treated FMP during storage were assessed, including hardness, the content, monosaccharide compositions, Mw distribution, and nanostructure of WSP, SSP, and CSP. Compared to TP treatment, HPP could effectively improve the texture quality of FMP by inhibiting pectin degradation during storage. All the findings presented in this study would help to provide new insights into regulating the texture quality of FMP.

Multi-omics in COVID-19: Seeing the unseen but overlooked in the clinic.

COVID-19 is an ongoing pandemic of global concern and is unlikely to disappear. This commentary discusses how multi-omics technologies have helped uncover the molecular processes and dynamics underlying COVID-19 initiation, progression, and transmission, and how lack of standardization has limited their application in clinical settings.

A prostate cancer tissue specific spectral library for targeted proteomic analysis.

Prostate cancer is the most common cancer in males worldwide. Mass spectrometry-based targeted proteomics has demonstrated great potential in quantifying proteins from formalin-fixed paraffin-embedded (FFPE) and (fresh) frozen biopsy tissues. Here we provide a comprehensive tissue-specific spectral library for targeted proteomic analysis of prostate tissue samples. Benign and malignant FFPE prostate tissue samples were processed into peptide samples by pressure cycling technology (PCT)-assisted sample preparation, and fractionated with high-pH reversed phase liquid chromatography (RPLC). Based on data-dependent acquisition (DDA) MS analysis using a TripleTOF 6600, we built a library containing 108,533 precursors, 84,198 peptides and 9384 unique proteins (1% FDR). The applicability of the library was demonstrated in prostate specimens.

Effect of hydrothermal-calcium chloride treatment on pectin characteristics and related quality in green peppers during storage.

Effects of hydrothermal (HT)-calcium chloride (CaCl2) treatment on pectin characteristics and related quality in green peppers during storage were assessed. The results showed that the changes of physicochemical quality in all green peppers were similar during storage. Weight loss percentage increased, firmness, the content of free water and bound water decreased during storage. Water-soluble pectin (WSP) notably increased, but sodium carbonate-soluble pectin (SSP) and chelate-soluble pectin (CSP) decreased. Galacturonic acid (GalUA), rhamnose (Rha), galactose (Gal), and arabinose (Ara) were the crucial compositions in the backbone and branched chains of pectin in green peppers. Rha and Gal increased, but Ara decreased in pectin after storage. The changes in the ratio of Rha/GalUA, Ara/Gal, and (Gal + Ara)/Rha represented that the backbone and branched chains of pectin in green peppers depolymerized to some extent after storage. Comparing with other green peppers, HT-CaCl2 treated green peppers posed lower weight loss percentage and WSP content, higher firmness, the content of free water, bound water, SSP, and CSP during storage. Otherwise, most pectin compositions in HT-CaCl2 treated green peppers showed high molar ratio after storage. Hence, HT-CaCl2 treatment was an effective way to retain pectin characteristics and related quality of green peppers, and further inhibited the softening of green peppers during storage.

Changes in pectin characteristics of jujube fruits cv "Dongzao" and "Jinsixiaozao" during cold storage.

Softening is one of the main factors affecting market value and consumer preferences for jujubes, and it was closely related to the modification and depolymerization of pectin. Changes in characteristics of three pectins (water-soluble pectin (WSP), sodium carbonate-soluble pectin (SSP) and chelate-soluble pectin (CSP)), including their contents, degree of methylesterification (DM), neutral sugar compositions, the molecular weight (Mw ) distributions and nanostructures, from two jujube fruits cv Dongzao (DZ) and Jinsixiaozao (JS) during cold storage were assessed. The results showed that variation in pectin characteristics during cold storage was similar between DZ and JS. The reduction of firmness corresponded to a conversion of water-insoluble pectin to WSP during cold storage. DM of WSP presented an increase trend in the late storage. Rhamnose (Rha), arabinose (Ara) and glucose (Glc) were the crucial compositions in three pectins, and most neutral sugar compositions in three pectins first increased and then decreased during cold storage. Changes in the ratio of (galactose (Gal)+Ara)/Rha and Ara/Gal represented that the branch chains of rhamnogalacturonan-I in three pectins depolymerized after storage. The high Mw in WSP and SSP of jujubes were solubilized and extensively depolymerized into pectin with lower Mw after storage. AFM images showed an increase in short chains and branch structures of three pectins after storage. Overall, three pectins in DZ and JS depolymerized and solubilized during cold storage. WSP and SSP were more contributed to the softening of jujubes compared to CSP, and they played the critical role for regulating the softening of jujube fruits during cold storage. PRACTICAL APPLICATION: Softening is one of the main factors affecting market value and consumer preferences for jujubes, and it was closely related to the modification and depolymerization of pectin. Changes in characteristics of three pectins (WSP, SSP, CSP), including their contents, degree of methylesterification, neutral sugar compositions, the molecular weight distributions and nanostructures, from two jujube fruits cv Dongzao (DZ) and Jinsixiaozao (JS) during cold storage were assessed. Three pectins in DZ and JS depolymerized and solubilized during cold storage. WSP and SSP were more contributed to the softening of jujubes compared to CSP, and they played the critical role for regulating the softening of jujube fruits during cold storage. This study would elucidate the mechanism of jujube softening and help to regulate the postharvest quality during cold storage.

Rehabilitation training combined acupuncture for limb hemiplegia caused by cerebral infarction: A protocol for a systematic review of randomized controlled trial.

BACKGROUND: Previous studies have reported that rehabilitation training combined acupuncture (RTA) can be used for the treatment of limb hemiplegia (LH) caused by cerebral infarction (CI). However, its effectiveness is still unclear. In this systematic review study, we aim to evaluate the effectiveness and safety of RTA for LH following CI. METHODS: We will retrieve the databases of CENTRAL, EMBASE, MEDILINE, CINAHL, AMED, CBM, PUBMED, and CNKI from inception to June 1, 2020 with no language restrictions. The randomized controlled trials of RTA for evaluating effectiveness and safety in patients with LH following CI will be included. Cochrane risk of bias tool will be used to measure the methodological quality for all included studies. Two authors will independently select the studies, extract the data, and assess the methodological quality of included studies. A third author will be invited to discuss if any disagreements exist between 2 authors. We will perform heterogeneity assessment before carrying out meta-analysis. According to the heterogeneity, we select random effect model or fixed effect model for meta-analysis of the included cohort studies. Cochrane risk of bias tool will be used to determine the methodological quality for included studies. RevMan 5.3 software (Cochrane Community, London, UK) will be utilized to perform statistical analysis. RESULTS: This systematic review will assess the effectiveness and safety of RTA for LH caused by CI. The primary outcome includes limbs function, as measured by the Wolf Motor Function Test (WMFT) Assessment scale, or other associated scales. The secondary outcomes consist of muscle strength, muscle tone, quality of life, and any adverse events. CONCLUSION: The findings of this study will summarize the current evidence of RTA for LH caused by CI, and may provide helpful evidence for the clinical treatment. DISSEMINATION AND ETHICS: The findings of this study are expected to be published in peer-reviewed journals. It does not require ethical approval, because no individual data will be utilized in this study. SYSTEMATIC REVIEW REGISTRATION: INPLASY202070114.

Generalized model for conic-V-shaped flexure hinges.

This paper presents a new class of flexure hinges, namely, conic-V-shaped flexure hinges (CFHs), which can be used as a generalized model for flexure hinges with profiles such as parabolic-V-shape, elliptical-V-shape, and hyperbolic-V-shape. Compliance and precision equations for the CFHs were derived as a set of nonlinear equations using Castigliano's second theorem. The parameters of the nonlinear equations inputted to the compliance and precision matrices were based on the generalized equations used for conic curves in polar coordinates. Furthermore, the compliance equations were verified by means of finite element analysis and experiments. The errors in the finite element and experimental results were within 10% and 8% compared to the analytical results, respectively. Finally, the effects of dimensional parameters on the analytical model could be effectively analyzed by numerical simulations and comparisons.

Changes in free amino acids of fermented minced peppers during natural and inoculated fermentation process based on HPLC-MS/MS.

Free amino acids (FAAs) participate in the synthesis of quality characteristic ingredients and taste substances in fermented minced peppers (FMPs), and they can be affected by fermentation method and time. In this study, changes in FAAs of FMP during natural fermentation (NF) and inoculated fermentation (IF) process were characterized by HPLC-MS/MS. The results showed that a total of 20 FAAs were identified, including 8 essential amino acids (AAs), 2 semiessential AAs, and 10 nonessential AAs. Comparing with other FAAs, Gln, Arg, and Asn presented higher content in the whole NF or IF periods. The FAAs content of NF and IF samples showed similar tendency during fermentation process, which first increased and then decreased. The highest content of most FAAs in IF and NF samples was obtained on the 18- and 12-day, respectively. Moreover, the FAA content in IF samples was higher than that in NF ones at the same fermentation time. The taste AAs content accounted for 23.3% to 50.0% in total FAAs, and taste activity value of bitter and umami was higher than sweet and aromatic ones. Based on principal component analysis, comprehensive analysis ranking, and heat map clustering analysis, FMP obtained by IF posed better FAAs quality than NF samples during fermentation process. PRACTICAL APPLICATION: Free amino acids (FAAs) participate in the synthesis of quality characteristic ingredients and taste substances in fermented minced peppers (FMPs), and they can be affected by fermentation method and time. Changes in FAA varieties and level in FMP during natural fermentation (NF) and inoculated fermentation (IF) process were analyzed using HPLC-MS/MS. Then, they were performed by principal component analysis (PCA), and clustered with the heat map. The results showed that FMP obtained by IF posed better FAAs quality than NF samples in the whole fermentation. HPLC-MS/MS was a reliable and effective mean for determining the FAAs and could provide regulation guidelines for improving the quality of FMP during fermentation process.

Post-Stroke Microglia Induce Sirtuin2 Expression to Suppress the Anti-inflammatory Function of Infiltrating Regulatory T Cells.

Ischemic stroke is among the leading causes of death and disability across the globe. Post-stroke neuroinflammation contributes to the pathophysiology of ischemic stroke in the acute phase through damaging neurons in the penumbra region. Infiltrating regulatory T cells (Treg cells) provide neuronal protection in ischemic brains. In the current study using a mouse-transient middle cerebral artery occlusion (MCAO) model, we characterized the changes of sirtuin expression in infiltrating Treg cells in the acute phase of ischemia. We found that Sirt2 was remarkably upregulated in infiltrating Treg cells at day 3 post-MCAO. In vitro inhibition of Sirt2 activity enhanced the expression of immunosuppression-associated molecules including forkhead box P3 (Foxp3) in Treg cells. Using a lentiviral system to express exogenous Sirt2 in Treg cells, we found that Sirt2 weakened the anti-inflammatory effect of Treg cells on pro-inflammatory macrophages. Additionally, post-MCAO microglia increased Sirt2 expression in Treg cells in a cell-to-cell contact manner. We further found that microglia remarkably induced hypoxia-inducible factor 1-alpha (HIF-1α) expression in Treg cells, and inhibition of HIF-1α abolished microglia-induced Sirt2 upregulation. Collectively, we discovered a novel mechanism by which the immunoregulatory activity of infiltrating Treg cells is modulated after ischemia.

Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK-related kinase.

Members of the MAPK superfamily are known as key regulators of ciliogenesis. Long flagellar (LF) 4, a MAPK-related kinase in Chlamydomonas, is the first kinase that was implicated in ciliary assembly and length. However, little is known about its cellular properties, regulation, and molecular functions. LF4 is localized both in the flagella and cell body with enrichment at the 2 basal bodies, shown by super-resolution microscopy. LF4 is constitutively phosphorylated at T159 at the kinase activation loop and remains at the basal bodies during flagellar assembly. Gene mutations that affect the kinase activity or T159 phosphorylation alter the localization of LF4 at the basal bodies, and the mutants fail to rescue lf4-3, a null mutant. LF4 does not affect the velocities of intraflagellar transport (IFT). However, LF4 null mutation induces accumulation of IFT proteins in the flagellum and reduces the phosphorylation of the kinesin-II subunit FLA8/KIF3B, indicating that LF4 negatively regulates IFT entry. Furthermore, LF2, a cell cycle-related kinase, and LF3, a novel protein, are required for LF4 phosphorylation. Our study demonstrates that LF4 is likely a constitutively active kinase that is regulated by LF2 and regulates IFT entry at the basal bodies to control flagellar assembly and length.-Wang, Y., Ren, Y., Pan, J. Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK-related kinase.

A novel role of Krüppel-like factor 4 in Zhikong scallop Chlamys farreri during spermatogenesis.

Krüppel-like factor 4 (KLF4) is a kind of zinc finger transcription factor, which is involved in terminal differentiation of epithelial cells and reprogramming of somatic cells to induced pluripotent stem (iPS) cells in mammals. In the present study, we identified a full-length cDNA of Klf4 in Zhikong scallop Chamys farreri (Cf-Klf4) and found that Cf-Klf4 presented a sexual dimorphic expression characteristic in C. farreri gonads. Cf-Klf4 expression was significantly higher in testes than in ovaries from growing stage to mature stage detected by quantitative real-time PCR, and was located in male gametes, except for spermatozoa during spermatogenesis through in situ hybridization and immunohistochemistry, while no positive signal was visible in female gametes during oogenesis. Furthermore, the knockdown of Cf-Klf4 in testes by means of in vivo RNA interference led to an obviously developmental retardance, lower gonadosomatic index, less male gametes and more apoptotic spermatocytes. Interestingly, we found that two out of eight scallops showed a hermaphroditic phenotype characteristic of male-to-female sex reversal when the Klf4 mRNA and protein levels were knocked down in males. These results verified that Klf4 plays an important role in testis functional maintenance and is necessary in spermatogenesis of C. farreri.

Enhancing cGMP in experimental progressive renal fibrosis: soluble guanylate cyclase stimulation vs. phosphodiesterase inhibition.

cGMP serves as the main second messenger of nitric oxide (NO). Antifibrotic effects of enhancing renal cGMP levels have recently been documented in experimental acute anti-Thy-1 glomerulonephritis. The present study compares the effects of the cGMP production-increasing soluble guanylate cyclase (sGC) stimulator BAY 41-2272 with those of the cGMP degradation-limiting phosphodiesterase inhibitor pentoxifylline (PTX) in a progressive model of renal fibrosis. At 1 wk after induction of anti-Thy-1-induced chronic glomerulosclerosis (cGS), rats were randomly assigned to groups as follows: cGS, cGS + BAY 41-2272 (10 mg x kg body wt(-1) x day(-1)), or cGS + PTX (50 mg x kg body wt(-1) x day(-1)). BAY 41-2272 and PTX reduced systolic blood pressure significantly. At 16 wk, tubulointerstitial expressions of sGC mRNA and NO-induced cGMP synthesis were increased in untreated cGS animals, whereas their glomerular activity was depressed compared with normal controls. Tubulointerstitial and glomerular cGMP production in response to NO were significantly enhanced in animals treated with BAY 41-2272, but not in those treated with PTX. BAY 41-2272 administration resulted in marked reductions of glomerular and tubulointerstitial histological matrix accumulation, expression of TGF-beta1 and fibronectin, macrophage infiltration, and cell proliferation as well as improved renal function. In contrast, only moderate and nonsignificant renoprotective changes were observed in the cGS + PTX group. In conclusion, increasing renal cGMP production through BAY 41-2272 significantly improved renal NO-cGMP signaling and limited progression in anti-Thy-1-induced chronic renal fibrosis, whereas inhibition of cGMP degradation by PTX was only moderately effective. The findings indicate that pharmacological enhancement of renal cGMP levels by sGC stimulation represents a novel and effective antifibrotic approach in progressive kidney disorders.

Stimulation of soluble guanylate cyclase slows progression in anti-thy1-induced chronic glomerulosclerosis.

BACKGROUND: A critical role of soluble guanylate cyclase and nitric oxide-dependent cyclic 3',5'-guanosine monophosphate (cGMP) production for glomerular matrix expansion has recently been documented in a rat model of acute anti-thy1 glomerulonephritis. The present study analyzes the renal activity of the nitric oxide-cGMP signaling cascade in and the effect of the specific soluble guanylate cyclase stimulator Bay 41-2272 on a progressive model of anti-thy1-induced chronic glomerulosclerosis. METHODS: Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized rats. One week after disease induction, animals were randomly assigned to chronic glomerulosclerosis, chronic glomerulosclerosis plus Bay 41-2272 (10 mg/kg body weight/day) or chronic glomerulosclerosis plus hydralazine (15 mg/kg body weight/day). In week 16, analysis included effects on systolic blood pressure, proteinuria, kidney function, glomerular and tubulointerstitial matrix protein accumulation, expression of transforming growth factor-beta1 (TGF-beta1), fibronectin and plasminogen activator inhibitor type 1 (PAI-1), macrophage infiltration, cell proliferation, basal and nitric oxide-stimulated cGMP production as well as tubulointerstitial mRNA expression of alpha 1 and beta 1 soluble guanylate cyclase. RESULTS: The moderately elevated systolic blood pressure seen in the chronic glomerulosclerosis group was comparably decreased by both treatments. Compared to normal controls, soluble guanylate cyclase mRNA expression and nitric oxide-stimulated cGMP production were up-regulated in the tubulointerstitium of the untreated chronic glomerulosclerosis animals, while its activity was decreased in glomeruli. Bay 41-2272 treatment enhanced glomerular and tubulointerstitial nitric oxide-cGMP signaling significantly. This went along with markedly reduced glomerular and tubulointerstitial macrophage infiltration, number of proliferating cells, matrix expression and accumulation, as well as improved kidney function. In contrast, hydralazine therapy did not significantly affect renal nitric oxide-cGMP signaling, macrophage number, cell proliferation, matrix protein expression and accumulation. CONCLUSION: Glomerular and tubulointerstitial soluble guanylate cyclase activity are discordantly altered in anti-thy1-induced chronic glomerulosclerosis. Stimulation of soluble guanylate cyclase signaling by Bay 41-2272 limits the progressive course of this model toward tubulointerstitial fibrosis and impaired renal function at least in part in a blood pressure-independent manner. The results suggest that soluble guanylate cyclase activation counteracts fibrosis and progression in chronic renal disease.