BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

BACKGROUND: An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-ß (transforming growth factor-ß) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-ß family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-ßR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-ß-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. METHODS: Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-ß signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. RESULTS: BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-ß signaling by downregulating TGF-ß expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. CONCLUSIONS: These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-ß signaling in PASMCs. Such rebalance of BMP/TGF-ß pathways is translationally important for PAH alleviation.

Mechanical, Thermal Properties, and Extreme Phase Stability of High-Entropy Diborides (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2.

High-entropy diborides (HEDBs) have gained significant attention in industrial applications due to their vast composition space and tunable properties. We propose a solid solution reaction at high temperatures and pressures that successfully synthesized and sintered a novel, dense, and phase-pure HEDB (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2. A high asymptotic Vickers hardness of 26.3 ± 0.6 GPa and a bulk modulus of 320.5 ± 10.6 GPa were obtained. Additionally, we investigated the thermal oxidation process using TG-DSC from room temperature to 1500 °C and explored the phase stability of HEDBs under high-pressure conditions through in situ high-pressure synchrotron radiation X-ray diffraction. We analyzed the formation of lattice distortion, chemical bonding, and band structure in (V0.2Nb0.2Ta0.2Cr0.2W0.2)B2 using first-principles calculations. Surprisingly, we found that the predominant distortion in diborides occurs in the boron layer, supported by ELF. This may be due to uneven electron transfer rather than a straightforward correlation with the atomic radius. These results provide a novel synthesis process and additional experimental data on the mechanical and thermal properties and high-pressure phase stability of HEDBs. Our study offers further insights into the microscopic structure of lattice distortion in HEDBs, which could prove crucial for the selection and design of engineering advanced HEDBs.

A Method Probing High-Temperature Oxidation Behavior of Crystalline Materials.

To date, the oxidation behavior of crystal materials is not fully understood; additional research is needed to understand the oxidation of materials. Herein, density functional theory (DFT) calculations and a 3D kinetic Monte Carlo (KMC) model are used to investigate the infiltration and diffusion behaviors of oxygen atoms within the crystal. Oxygen molecules readily adsorbes on crystal surfaces of the material and rapidly dissociates, verified by both first-principles calculations and energy-dispersive spectrometer (EDS) results. The infiltration ability of oxygen atoms into the inner crystal layers is affected by the surrounding oxygen atom, lattice compactness, and other factors. Energy-barrier calculations show that crystal thin/dense layers have significant effects on the crystal oxidation process, so high-pressure technology is used to investigate this correlation experimentally. KMC calculations and thermogravimetric analyses (TGA) show the infiltration behavior of oxygen atoms in the main crystal plane (211) toward the inner layers has the highest proportion to the actual high-temperature oxidation behavior of the title material. The results of both the KMC calculations and thermal experiments show the material peeled off upon further oxidation, which accelerates oxidation. At the same time, high-pressure treatment increases the oxidation resistance of materials at lower temperatures (<600 °C).

Kinetin inhibits hepatic stellate cell activation and induces apoptosis via interactions with the TGF-ß1/Smad signaling pathway.

Hepatic fibrosis is the pathological repair response of the liver to chronic injury; hepatic stellate cell (HSC) activation is the central link in the pathogenesis of hepatic fibrosis. Previously, we showed that kinetin, a plant cytokinin hormone, has a protective effect on CCl4-induced liver injury in mice. However, the role of kinetin in liver fibrosis remains unclear. We aimed to study these protective effects and to determine the mechanisms by which kinetin mediates HSC activation and apoptosis. For this purpose, the human HSC line LX-2 was treated with 10 ng/ml transforming growth factor-ß1 (TGF-ß1) for 24 h to stimulate activation. We found that treatment with kinetin at the sub-cytotoxic dose of 40 µg/ml for 48 h reduced the expression of the HSC activation marker α-SMA and inhibited the secretion of extracellular matrix proteins. In addition, kinetin was found to inhibit the proliferation and migration of LX-2 cells. We found that kinetin induced apoptosis in LX-2 cells by increasing the level of cleaved-caspase 3 and the Bax-to-Bcl-2 ratio. Interestingly, these effect were not observed in quiescent HSCs, suggesting that they are activation-dependent. Further study showed that kinetin attenuates activation and promotes apoptosis of LX-2 cells in vitro in part by suppressing the TGF-ß1/Smad signaling pathway.

Detection of aberrant DNA methylation patterns in sperm of male recurrent spontaneous abortion patients.

Aberrant DNA methylation patterns in sperm are a cause of embryonic failure and infertility, and could be a critical factor contributing to male recurrent spontaneous abortion (RSA). The purpose of this study was to reveal the potential effects of sperm DNA methylation levels in patients with male RSA. We compared sperm samples collected from fertile men and oligoasthenospermia patients. Differentially methylated sequences were identified by reduced representation bisulfite sequencing (RRBS) methods. The DNA methylation levels of the two groups were compared and qRT-PCR was used to validate the expression of genes showing differential methylation. The results indicated that no difference in base distribution was observed between the normal group and the patient group. However, the chromosome methylation in these two groups was markedly different. One site was located on chromosome 8 and measured 150 bp, while the other sites were on chromosomes 9, 10, and X and measured 135 bp, 68 bp, and 136 bp, respectively. In particular, two genes were found to be hypermethylated in these patients, one gene was DYDC2 (placed in the differential methylation region of chromosome 10), and the other gene was NXF3 (located on chromosome X). Expression levels of DYDC2 and NXF3 in the RSA group were significantly lower than those in the normal group (P < 0.05). Collectively, these results demonstrated that changes in DNA methylation might be related to male RSA. Our findings provide important information regarding the potential role of sperm DNA methylation in human development.

Hemodynamic effect through a novel endoscopic intervention in management of varices and hypersplenism (with video).

BACKGROUND AND AIMS: We previously reported a new and combined EUS-guided intervention in a patient with portal hypertension, consisting of obliteration of varices and partial splenic embolization (PSE). Performing PSE is known to diminish the increase in portal venous pressure after endoscopic intervention for varices. The aim of this study was to use multidetector CT portal venography to evaluate the anatomy of esophagogastric varices (EGV) and the impact on hemodynamics of portosystemic collaterals shortly after the concomitant procedures. METHODS: From October 2019 to December 2020, 5 patients with cirrhosis and with clinically significant portal hypertension who had variceal bleeding history and hypersplenism were treated with combined endoscopic obliteration for varices and EUS-guided PSE. Multidetector CT portal venography was applied to assess the anatomic drainage patterns of the EGV, diameters of feeders and drainage vessels, and splenic embolization rate. RESULTS: Within 5 days after concomitant endoscopic interventions, we observed decreased mean diameters of the left gastric vein, short gastric vein, and azygos vein as .3 mm, 1.0 mm, and 5.2 mm compared with 3.11 mm, 7.1 mm, and 5.4 mm before the procedures, respectively. Patients showed increased white blood cells (mean count of 2.7 × 109/L before vs 5.8 × 109/L after) and platelets (mean count of 52.8 × 109/L before vs 95.8 × 109/L after). The mean splenic embolization rate was 64.5% (range, 28.8%-84.6%). CONCLUSIONS: Our experience may illustrate an alternative technique of combining EUS-guided PSE with endoscopic therapy of varices to treat patients with portal hypertension.

Pressure-Induced Phase Transition and Compression Properties of HfO2 Nanocrystals.

Nanoparticles exhibit unique properties due to their surface effects and small size, and their behavior at high pressures has attracted widespread attention in recent years. Herein, a series of in situ high-pressure X-ray diffraction measurements with a synchrotron radiation source and Raman scattering have been performed on HfO2 nanocrystals (NC-HfO2) with different grain sizes using a symmetric diamond anvil cell at ambient temperature. The experimental data reveal that the structural stability, phase transition behavior, and equation of state for HfO2 have an interesting size effect under high pressure. NC-HfO2 quenched to normal pressure is characterized by transmission electron microscopy to determine the changing behavior of grain size during phase transition. We found that the rotation of the nanocrystalline HfO2 grains causes a large strain, resulting in the retention of part of an orthorhombic I (OI) phase in the sample quenched to atmospheric pressure. Furthermore, the physical mechanism of the phase transition of NC-HfO2 under high pressure can be well explained by the first-principles calculations. The calculations demonstrate that NC-HfO2 has a strong surface effect, that is, the surface energy and surface stress can stabilize the structures. These studies may offer new insights into the understanding of the physical behavior of nanocrystal materials under high pressure and provide practical guidance for their realization in industrial applications.

Insights into the Bond Behavior and Mechanical Properties of Hafnium Carbide under High Pressure and High Temperature.

Hafnium carbide (HfC) is a potential candidate of ultrahigh-temperature ceramics (UHTCs) and has attracted significantly widespread interest in recent years. Here, we have synthesized high-purity HfC samples with NaCl-type structure by using a high-pressure solid-solid reaction. The structural stability, equation of state, plastic deformation, yield strength, and bonding properties under high pressure are investigated by a series of in situ high-pressure synchrotron-radiation angle-dispersive X-ray diffraction experiments combined with first-principles calculations. The yield strength of HfC (∼18 GPa) is obtained from analyzing the plastic deformation behavior under high pressure. In addition, we have successfully prepared bulk HfC ceramics with high density using a high-pressure and high-temperature method. The synthesized sample possesses a desirable Vickers hardness of 24.2 GPa and an excellent fracture toughness of 5.0 MPa·m1/2. The present results offer insights into the achievable application of HfC ceramics under extreme conditions and provide a powerful guide for the further design and synthesis of other high-performance UHTCs.

aMAP risk score predicts hepatocellular carcinoma development in patients with chronic hepatitis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with chronic hepatitis. In this international collaboration, we sought to develop a global universal HCC risk score to predict the HCC development for patients with chronic hepatitis. METHODS: A total of 17,374 patients, comprising 10,578 treated Asian patients with chronic hepatitis B (CHB), 2,510 treated Caucasian patients with CHB, 3,566 treated patients with hepatitis C virus (including 2,489 patients with cirrhosis achieving a sustained virological response) and 720 patients with non-viral hepatitis (NVH) from 11 international prospective observational cohorts or randomised controlled trials, were divided into a training cohort (3,688 Asian patients with CHB) and 9 validation cohorts with different aetiologies and ethnicities (n = 13,686). RESULTS: We developed an HCC risk score, called the aMAP score (ranging from 0 to 100), that involves only age, male, albumin-bilirubin and platelets. This metric performed excellently in assessing HCC risk not only in patients with hepatitis of different aetiologies, but also in those with different ethnicities (C-index: 0.82-0.87). Cut-off values of 50 and 60 were best for discriminating HCC risk. The 3- or 5-year cumulative incidences of HCC were 0-0.8%, 1.5-4.8%, and 8.1-19.9% in the low- (n = 7,413, 43.6%), medium- (n = 6,529, 38.4%), and high-risk (n = 3,044, 17.9%) groups, respectively. The cut-off value of 50 was associated with a sensitivity of 85.7-100% and a negative predictive value of 99.3-100%. The cut-off value of 60 resulted in a specificity of 56.6-95.8% and a positive predictive value of 6.6-15.7%. CONCLUSIONS: This objective, simple, reliable risk score based on 5 common parameters accurately predicted HCC development, regardless of aetiology and ethnicity, which could help to establish a risk score-guided HCC surveillance strategy worldwide. LAY SUMMARY: In this international collaboration, we developed and externally validated a simple, objective and accurate prognostic tool (called the aMAP score), that involves only age, male, albumin-bilirubin and platelets. The aMAP score (ranged from 0 to 100) satisfactorily predicted the risk of hepatocellular carcinoma (HCC) development among over 17,000 patients with viral and non-viral hepatitis from 11 global prospective studies. Our findings show that the aMAP score had excellent discrimination and calibration in assessing the 5-year HCC risk among all the cohorts irrespective of aetiology and ethnicity.

Exploring Physical Properties of Tantalum Carbide at High Pressure and Temperature.

Among the transition metal carbides, tantalum carbide (TaC) has gained significant interest due to its attractive mechanical and electronic properties. Here, we have performed high pressure and high temperature (HPHT) measurements on the physical properties of TaC under 5.5 GPa and rhythmically increased temperatures from 1000 to 1500 °C. The microscopic deviatoric strain, Vickers hardness, fracture toughness, grain size, and microstructures are characterized by X-ray diffraction (XRD), scanning electronic microscopy (SEM), transmission electron microscopy (TEM), and microhardness tests. The results reveal that the HPHT sintering causes the densification, which increases the mechanical properties of TaC. At 5.5 GPa and 1300 °C, the Vickers hardness, fracture toughness, relative density, and Young's modulus of TaC are 21.0 GPa, 7.4 MPa m1/2, 457 GPa, and 97.7%, respectively, which are in good agreement with available experimental and theoretical values. It is found that the mechanical properties of TaC are highly impressible to the microstructures and microscopic deviatoric stress. Our cadent HPHT sintering technique will provide powerful guidance for further synthesis and design of other novel ultrahigh temperature ceramics (UHTCs).

Mechanical properties of tantalum carbide from high-pressure/high-temperature synthesis and first-principles calculations.

As a member of the refractory metal carbide family of materials, TaC is a promising candidate for ultra-high temperature ceramics (UHTC) with desirable mechanical strength. TaC sample quality and therefore mechanical properties are strongly dependent on synthesis method, and atomistic origins of mechanical failure are difficult to assign. Here, we have successfully synthesized high quality densified TaC samples at 5.5 GPa and 1400 °C using the high pressure and high temperature (HPHT) sintering method, with Vickers hardness determined to be 20.9 GPa. First-principles calculations based on the recently developed strain-stress method show that the ideal indentation strength of TaC is about 23.3 GPa in the (11[combining macron]0)[001] direction, in excellent agreement with experimental results. The detailed indentation shear deformation analysis and structural snapshots from the calculations indicate that the slip dislocations of TaC layers are the main structural deformation mode during the Vickers indentation process, and that the strong directional Ta-C bonds are responsible for the high mechanical strength of TaC. HPHT synthesis is shown to produce TaC samples with superior strength, and together with accurate first-principles calculations offers crucial insights for rational design and synthesis of novel and advanced UHTC materials.

Early identification of carotid vulnerable plaque in asymptomatic patients.

BACKGROUND: This study was to explore the influencing factors of atherosclerotic plaque formation and stability in patients with asymptomatic carotid atherosclerotic plaques, so as to identify the vulnerable plaques at early stage, and then find high-risk group of cardio-cerebrovascular events for early clinical intervention to reduce related mortality and disability. METHODS: A total of 302 enrolled patients with asymptomatic carotid atherosclerotic plaques were divided into 3 groups based on the results of carotid artery color Doppler ultrasound: atherosclerotic unstable plaque (UP) group, atherosclerotic stable plaque (SP) group, and control group without plaques. Serum markers were measured by ELISA. χ2 test, t test, Pearson correlation analysis, and Logistic multivariate regression analysis were used in the analysis, and P < 0.05 was considered statistically significant. RESULTS: It revealed that high MMP-9, LOX-1and YKL-40 were independent risk factors for unstable plaque formation. The area under the curve (AUC) of serum markers combined with MMP-9, LOX-1 and YKL-40 was 0.850, with sensitivity 87.67%, specificity 81.13%, and diagnostic accuracy 84.92%, which was significantly better than the individual diagnostic efficacy of other three factors. The accuracy rate of Crouse Plaque Score (CPS) in the diagnosis of vulnerable plaques was 61.90%, the 10-year ICVD diagnosis accuracy rate was 56.75%, and the diagnostic accuracy of serum markers was significantly better than CPS and 10-year ICVD. CONCLUSION: Noninvasive cervical color Doppler ultrasound combined with serum markers MMP-9, LOX-1 and YKL-40 have significant early recognition effect on asymptomatic carotid vulnerable plaque patients.

O-Linked ß-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

OBJECTIVE: Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked ß-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. APPROACH AND RESULTS: In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. CONCLUSIONS: These results suggest that O-GlcNAcylation of A20 plays a key role in the negative regulation of NF-κB signaling cascades in TNF-α-treated vascular smooth muscle cells in culture and in acutely injured arteries, thus protecting against inflammation-induced vascular injury.

Elongin B is a binding partner of the male germ cell nuclear speckle protein sperm-associated antigen 16S (SPAG16S) and is regulated post-transcriptionally in the testis.

In this study we identified Elongin B, a regulatory subunit of the trimeric elongation factor Elongin ABC, which increases the overall rate of elongation by RNA polymerase II, as a major binding partner of sperm-associated antigen 16S (SPAG16S), a component of nuclear speckles. Nuclear speckles are nuclear subcompartments involved in RNA maturation. Previously, we showed that SPAG16S is essential for spermatogenesis. In the present study, a specific antibody against mouse Elongin B was generated and reacted with a protein with the predicted size of Elongin B in the testis; immunofluorescence staining revealed that the Elongin B was located in the nuclei and residual bodies. In round spermatids, Elongin B was colocalised with splicing factor SC35 (SC35), a marker of nuclear speckles. During the first wave of spermatogenesis, Elongin B transcripts were initially detected at Postnatal Day (PND) 8, and levels were greatly increased afterwards. However, Elongin B protein was only found from PND30, when germ cells progressed through spermiogenesis. Polysomal gradient analysis of Elongin B transcripts isolated from adult mouse testes revealed that most of the Elongin B mRNA was associated with translationally inactive, non-polysomal ribonucleoproteins. An RNA electrophoretic mobility shift assay demonstrated that the 3' untranslated region of the Elongin B transcript was bound by proteins present in testis but not liver extracts. These findings suggest that post-transcriptional regulation of Elongin B occurs in the testis, which is a common phenomenon during male germ cell development. As a major binding partner of SPAG16S, Elongin B may play an important role in spermatogenesis by modulating RNA maturation.

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8-iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8-iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the "9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27.

A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella.

A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.

Triptolide attenuates pressure overload-induced myocardial remodeling in mice via the inhibition of NLRP3 inflammasome expression.

Triptolide is the predominant active component of the Chinese herb Tripterygium wilfordii Hook F (TwHF) that has been widely used to treat several chronic inflammatory diseases due to its immunosuppressive, anti-inflammatory, and anti-proliferative properties. In the present study, we elucidated the cardioprotective effects of triptolide against cardiac dysfunction and myocardial remodeling in chronic pressure-overloaded hearts. Furthermore, the potential mechanisms of triptolide were investigated. For this purpose, C57/BL6 mice were anesthetized and subjected to transverse aortic constriction (TAC) or sham operation. Six weeks after the operation, all mice were randomly divided into 4 groups: sham-operated with vehicle group, TAC with vehicle group, and TAC with triptolide (20 or 100 µg/kg/day intraperitoneal injection) groups. Our data showed that the levels of NLRP3 inflammasome were significantly increased in the TAC group and were associated with increased inflammatory mediators and profibrotic factor production, resulting in myocardial fibrosis, cardiomyocyte hypertrophy, and impaired cardiac function. Triptolide treatment attenuated TAC-induced myocardial remodeling, improved cardiac diastolic and systolic function, inhibited the NLRP3 inflammasome and downstream inflammatory mediators (IL-1ß, IL-18, MCP-1, VCAM-1), activated the profibrotic TGF-ß1 pathway, and suppressed macrophage infiltration in a dose-dependent manner. Our study demonstrated that the protective effect of triptolide against pressure overload in the heart may act by inhibiting the NLRP3 inflammasome-induced inflammatory response and activating the profibrotic pathway.

IFT25, an intraflagellar transporter protein dispensable for ciliogenesis in somatic cells, is essential for sperm flagella formation.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the "9+2" microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice.

Smad3-mSin3A-HDAC1 Complex is Required for TGF-ß1-Induced Transcriptional Inhibition of PPARγ in Mouse Cardiac Fibroblasts.

BACKGROUND: We have recently demonstrated that activated transforming growth factor-ß (TGF-ß) signaling suppresses myocardial peroxisome proliferator-activated receptor γ (PPARγ) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-ß-induced PPARγ transcriptional inhibition. METHODS: Adult mouse cardiac fibroblasts were isolated and cultured. PPARγ promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified. RESULTS: In cultured cardiac fibroblasts transfected with a plasmid containing a human PPARγ promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-ß1-induced inhibition of PPARγ promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPARγ gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPARγ gene in cardiac fibroblasts in response to TGF-ß1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-ß-induced transcriptional suppression of PPARγ. CONCLUSION: These results suggest that TGF-ß1-induced inhibition of PPARγ transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPARγ promoter.