Inter-rater variability and repeatability in the assessment of the Tanner-Whitehouse classification of hand radiographs for the estimation of bone age.

OBJECTIVE: To determine which bones and which grades had the highest inter-rater variability when employing the Tanner-Whitehouse (T-W) method. MATERIALS AND METHODS: Twenty-four radiologists were recruited and trained in the T-W classification of skeletal development. The consistency and skill of the radiologists in determining bone development status were assessed using 20 pediatric hand radiographs of children aged 1 to 18 years old. Four radiologists had a poor concordance rate and were excluded. The remaining 20 radiologists undertook a repeat reading of the radiographs, and their results were analyzed by comparing them with the mean assessment of two senior experts as the reference standard. Concordance rate, scoring, and Kendall's W were calculated to evaluate accuracy and consistency. RESULTS: Both the radius, ulna, and short finger (RUS) system (Kendall's W = 0.833) and the carpal (C) system (Kendall's W = 0.944) had excellent consistency, with the RUS system outperforming the C system in terms of scores. The repeatability analysis showed that the second rating test, performed after 2 months of further bone age assessment (BAA) practice, was more consistent and accurate than the first. The capitate had the lowest average concordance rate and scoring, as well as the lowest overall concordance rate for its D classification. Moreover, the G classifications of the seven carpal bones all had a concordance rate less than 0.6. The bones with lower Kendall's W were likewise those with lower scores and concordance rates. CONCLUSION: The D grade of the capitate showed the highest variation, and the use of the Tanner-Whitehouse 3rd edition (T-W3) to determine bone age (BA) was frequently inconsistent. A more comprehensive description with a focus on inaccuracy bones or ratings and a modification to the T-W3 approach would significantly advance BAA.

PTOV1-AS1 desensitizes colorectal cancer cells to 5-FU through depressing miR-149-5p to activate the positive feedback loop with Wnt/ß-catenin pathway.

5-Fluorouracil is a commonly used chemotherapy drug for colorectal cancer. Resistance to 5-Fluorouracil remains a challenge. This research aimed to explore the mechanism of 5-Fluorouracil resistance in colorectal cancer. RT-qPCR and Western blot were used to determine the RNA and protein expression in both cells and exosome. Assays in vitro and in vivo were performed to measure the role of miR-149-5p in colorectal cancer cells. RIP, luciferase activity report, and RNA pulldown assay were applied to detect the association of PTOV1-AS1, SUV39H1, miR-149-5p, and FOXM1. MiR-149-5p was down-expressed in 5-Fluorouracil-resistant cells. MiR-149-5p enhanced the effectiveness of 5-Fluorouracil both in vitro and in vivo. Sensitive colorectal cancer cells released exosomal miR-149-5p to sensitize resistant cells to chemotherapy. Mechanistically, miR-149-5p targeted the FOXM1 to inactivate Wnt/ß-catenin pathway, and PTOV1-AS1 recruited SUV39H1 to suppress miR-149-5p transcription, in turn activating Wnt/ß-catenin pathway, and forming a positive feedback loop with FOXM1. PTOV1-AS1 inhibits miR-149-5p by a positive feedback loop with FOXM1-mediated Wnt/ß-catenin pathway, which provides insights into a potential novel target for enhancing the effectiveness of chemotherapy in colorectal cancer patients.

Dynamic viral integration patterns actively participate in the progression of BK polyomavirus-associated diseases after renal transplantation.

The classical lytic infection theory along with large T antigen-mediated oncogenesis cannot explain the BK polyomavirus (BKPyV)-associated tumor secondary to BKPyV-associated nephropathy (BKVAN), viremia/DNAemia, and viruria after renal transplantation. This study performed virome capture sequencing and pathological examination on regularly collected urine sediment and peripheral blood samples, and BKVAN and tumor biopsy tissues of 20 patients with BKPyV-associated diseases of different stages. In the early noncancerous stages, well-amplified integration sites were visualized by in situ polymerase chain reaction, simultaneously with BKPyV inclusion bodies and capsid protein expression. The integration intensity, the proportion of microhomology-mediated end-joining integration, and host PARP-1 and POLQ gene expression levels increased with disease progression. Furthermore, multiomics analysis was performed on BKPyV-associated urothelial carcinoma tissues, identifying tandem-like structures of BKPyV integration using long-read genome sequencing. The carcinogenicity of BKPyV integration was proven to disturb host gene expression and increase viral oncoprotein expression. Fallible DNA double-strand break repair pathways were significantly activated in the parenchyma of BKPyV-associated tumors. Olaparib showed an antitumor activity dose-response effect in the tumor organoids without BRCA1/2 genes mutation. In conclusion, the dynamic viral integration patterns actively participate in the progression of BKPyV-associated diseases and thus could be a potential target for disease monitoring and intervention.

Ductular reaction promotes intrahepatic angiogenesis through Slit2-Roundabout 1 signaling.

BACKGROUND AND AIMS: Ductular reaction (DR) expands in chronic liver diseases and correlates with disease severity. Besides its potential role in liver regeneration, DR plays a role in the wound-healing response of the liver, promoting periductular fibrosis and inflammatory cell recruitment. However, there is no information regarding its role in intrahepatic angiogenesis. In the current study we investigated the potential contribution of DR cells to hepatic vascular remodeling during chronic liver disease. APPROACH AND RESULTS: In mouse models of liver injury, DR cells express genes involved in angiogenesis. Among angiogenesis-related genes, the expression of Slit2 and its receptor Roundabout 1 (Robo1) was localized in DR cells and neoangiogenic vessels, respectively. The angiogenic role of the Slit2-Robo1 pathway in chronic liver disease was confirmed in ROBO1/2-/+ mice treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine, which displayed reduced intrahepatic neovascular density compared to wild-type mice. However, ROBO1/2 deficiency did not affect angiogenesis in partial hepatectomy. In patients with advanced alcohol-associated disease, angiogenesis was associated with DR, and up-regulation of SLIT2-ROBO1 correlated with DR and disease severity. In vitro, human liver-derived organoids produced SLIT2 and induced tube formation of endothelial cells. CONCLUSIONS: Overall, our data indicate that DR expansion promotes angiogenesis through the Slit2-Robo1 pathway and recognize DR cells as key players in the liver wound-healing response.

Greater bone marrow fat and myosteatosis are associated with lower vBMD but not asymptomatic vertebral fracture.

OBJECTIVES: Organ fat may affect bone metabolism and be associated with vertebral fracture (VF). This study aimed to explore relationships between VF, adiposity indexes measured by MRI, and volumetric BMD (vBMD) measured by quantitative CT (QCT). METHODS: Four hundred volunteers, ranging in age from 22 to 83 years, were recruited and underwent same-day abdominal QCT and chemical shift-encoded (CSE) MRI. We used MRI to quantify the fat content of bone marrow (BMF), psoas major and paraspinal muscles, and the liver. Abdominal fat, VF, and vBMD of the lumbar spine were measured by QCT. For VF discrimination analysis, we examined both the whole cohort (60 VF cases in 30 men and 30 women) and a restricted subgroup of those aged over 50 years (50 VF cases in 23 men and 27 women). RESULTS: Amongst the men, a 1 SD increase in BMF was associated with a 27.67 (95% CI, -32.71 to -22.62) mg/cm3 decrease in vBMD after adjusting for age and BMI. Amongst women, all adiposity indexes except for liver fat were significantly associated with vBMD, with BMF having the strongest association (ß, -24.00; 95% CI, -28.54 to -19.46 mg/cm3). Similar findings were also observed in participants aged over 50 years. The associations of adiposity indexes with vertebral fracture were not significant after adjusting for age in both sexes aged over 50 years. CONCLUSIONS: In both sexes, higher bone marrow fat was associated with lower vBMD at the spine. However, marrow fat and other adipose tissues were not associated with radiographic-based prevalent vertebral fractures. KEY POINTS: • In both sexes, higher bone marrow fat was associated with lower vBMD at the spine. • Among women, all adiposity indexes except for liver fat content were significantly associated with vBMD, with bone marrow fat having the strongest association. • Marrow fat and other adipose tissues were not associated with radiographic-based asymptomatic vertebral fractures.

The association of lumbar disc degeneration with lumbar vertebral trabecular volumetric bone mineral density in an urban population of young and middle-aged community-dwelling Chinese adults: a cross-sectional study.

INTRODUCTION: Lumbar intervertebral disc degeneration (LDD) and osteoporosis (OP) are age-related conditions that induce low back pain and have an impact on quality of life. The relationship between LDD and changes in bone mineral density (BMD) is, however, contentious and ever-changing. The purpose of this study is to investigate the relationship between lumbar vertebral volumetric BMD (vBMD) and LDD in an urban population of young and middle-aged community-dwelling Chinese adults. MATERIALS AND METHODS: 719 participants were recruited from among the subjects enrolled in a 10-year longitudinal study of degeneration of the spine and knee being conducted at the Beijing Jishuitan Hospital. The severity of LDD was graded using the five-grade Pfirrmann classification, and lumbar vertebral vBMD was measured using quantitative computed tomography (QCT). The relationship between the grade of intervertebral disc degeneration and lumbar vertebral vBMD was analyzed, and multiple linear regression was performed to adjust for covariates. RESULTS: The mean lumbar vBMD decreased as the grade of LDD increased (171.5 g/cm3, 147.8 g/cm3, and 124.3 g/cm3, respectively; P < 0.001). After adjusting for age, a higher LDD stage was associated with a lower mean L2-L4 vBMD, although a statistically significant correlation was observed only in men (standardized coefficient ß = - 0.656, P = 0.004). In men, there was a negative correlation between single-vertebra vBMD and degeneration of adjacent intervertebral discs, particularly those involving the L3 vertebra (L2-3 disc: ß = - 0.333, P < 0.001, L3-4 disc: ß = - 0.398, P < 0.001), as well as the mean grade of the L2-4 discs (ß = - 0.448, P < 0.001). However, the L5-S1 disc had a smaller correlation with age than others, and no statistically significant associations with lumbar vBMD were observed in either men (ß = - 0.024, P = 0.729) or women (ß = - 0.057, P = 0.396). CONCLUSION: Our study found that the degree of LDD was negatively associated with lumbar trabecular vBMD, although (excepting the L5-S1 disc), the relationship was statistically significant only in men.

The lumbar spinal endplate lesions grades and association with lumbar disc disorders, and lumbar bone mineral density in a middle-young general Chinese population.

BACKGROUND: Lumbar vertebral endplates lesions (LEPLs), one of the etiologies of low back pain (LBP), are one of the most prevalent causes of health-care costs. Despite progressively becoming the focus in recent years, almost all studies have concentrated on symptomatic patients rather than general populations. As a result, our study was designed to determine the prevalence and distribution patterns of LEPLs in a middle-young general population, as well as their associations with lumbar disc herniation (LDH), lumbar disc degeneration (LDD), and lumbar vertebral volumetric bone mineral density (vBMD). METHODS: Seven hundred fifty-four participants aged 20-60 years were recruited from the subjects enrolled in a 10-year longitudinal study of degeneration of the spine and knee being conducted at the Beijing Jishuitan Hospital and 4 of them were excluded due to the missing of MRIs. In this observational study, a lumbar quantitative computed tomography (QCT) and MRI scan were performed among participants within 48 h. T2-weighted sagittal lumbar MRI images for all included subjects were identified for LEPLs by two independent observers based on morphological and local characteristics. Lumbar vertebral vBMD was measured with QCT. The age, BMI, waistline, hipline, lumbar vBMD, LDD, and LDH were measured to investigate their associations with LEPLs. RESULTS: The prevalence of LEPLs was higher among the male subjects. 80% of endplates were recognition as no lesions with a substantial disparity between female (75.6%) and male subjects (83.4%) (p < 0.001). The most common lesions were "wavy/irregular" and "notched", and "fracture" is most involved in L3-4 inferior endplate both in two genders. LEPLs were found to be associated with LDH (≥ 2 levels: OR = 6.859, P < 0.001; 1 level: OR = 2.328, P = 0.002 in men. OR = 5.004, P < 0.001; OR = 1.805, P = 0.014 in women) reference for non-LDH, and hipline in men (OR = 1.123, P < 0.001). CONCLUSIONS: LEPLs are the common findings on lumbar MRIs in general population, particularly in men. The presence of these lesions and advance from slightly to severely could be mainly attributed to LDH and men's higher hipline.

Efficacy of autologous platelet-rich plasma for long-term glucocorticoids caused chronic wound: a case report.

The repair of bone explore wounds is one of the difficult problems in plastic and reconstruction surgery. Platelet-rich plasma (PRP) is a safe and efficient therapeutic option for various trauma, including Osteoarticular, musculoskeletal, and Wound injuries. However, the preparation and storage of PRP becomes challenging for patients with poor systemic status and requiring multi-use of PRP. The availability of safe, reliable tissue bank makes it possible. We report a case of a 42-year-old woman patient with a chronic hip wound combined with ischium bone exploration. And the patient who was treated with long-term glucocorticoids for rheumatoid arthritis has been through the experience of extensive conservative management. Thereafter necrosectomy and Vacuum-Assisted Closure (VAC) surgical procedure failed, and a PRP daily injection was performed at the ischial muscle and soft tissue. Neo-muscle appeared around the explored ischium bone after 8 weeks of injection and Complete wound healing was obtained in 3 months.

POTEE promotes colorectal carcinoma progression via activating the Rac1/Cdc42 pathway.

Current studies have shown that POTE ankyrin domain family members have high expressions as tumor antigens in malignant tumors, such as prostate cancer, ovarian cancer, breast cancer and the like. POTEE is a member of the POTE anchor protein family E. However, its role in colorectal carcinoma (CRC) has not been studied. In this study, the function of POTEE in CRC was examined for the first time and its correlation with CRC cell biological behaviors was analyzed. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry revealed that POTEE was remarkably overexpressed in CRC and associated with an aggressive phenotype. We also found that POTEE was localized in the cytoplasm. In addition, downregulation of POTEE expression can notably inhibit the proliferation, migration, and invasion of CRC cell in vitro, and repressed tumor growth and metastasis in vivo. In contrast, overexpression of POTEE could promote the aggressive behaviors of CRC cells. Mechanistically, POTEE promoted CRC migration, invasion and epithelial-mesenchymal transition (EMT) by increasing the activation of Rac1 and Cdc42. To summarize, these results suggested that POTEE might serve as an oncogene for CRC tumorigenesis and progression, and may become a novel molecular marker for clinical diagnosis and treatment.

Latissimus Dorsi-Myocutaneous Flap in the Repair of Titanium Mesh Exposure and Scalp Defect After Cranioplasty.

Titanium mesh was widely used for cranium defect repair but associated with high complication rates. In this study, the authors describe a method using latissimus dorsi-myocutaneous flap in the repair of titanium mesh exposure and scalp defect after cranioplasty, and the plate retaining is also achieved. Fifteen patients from April 2012 to May 2016 underwent this procedure, the age ranged from 32 to 62 years and 47 years old on average, and all the patient had plate exposure combined with surgical site infection and variation of scalp defect. All the patients had fully flap survive, and follow up ranged from 6 months to 24 months, 1 patient had titanium mesh re-expose and received additional operation to remove the plate. The free latissimus dorsi musculocutaneous flap could supply large size of bulky tissue coverage with good blood supply and strong anti-infection ability. This method was an option for retaining the titanium mesh and repairing the exposure for the mild infection with small size scalp defect patient.

Induction of intestinal stem cells by R-spondin 1 and Slit2 augments chemoradioprotection.

Cancer research has been rightly and successfully focused on prevention, early detection, and identification of specific molecular targets that distinguish the malignant cells from the neighbouring benign cells. However, reducing lethal tissue injury caused by intensive chemoradiotherapy during treatment of late-stage metastatic cancers remains a key clinical challenge. Here we tested whether the induction of adult stem cells could repair chemoradiation-induced tissue injury and prolong overall survival in mice. We found that intestinal stem cells (ISCs) expressed Slit2 and its single-span transmembrane cell-surface receptor roundabout 1 (Robo1). Partial genetic deletion of Robo1 decreased ISC numbers and caused villus hypotrophy, whereas a Slit2 transgene increased ISC numbers and triggered villus hypertrophy. During lethal dosages of chemoradiation, administering a short pulse of R-spondin 1 (Rspo1; a Wnt agonist) plus Slit2 reduced ISC loss, mitigated gut impairment and protected animals from death, without concomitantly decreasing tumour sensitivity to chemotherapy. Therefore Rspo1 and Slit2 may act as therapeutic adjuvants to enhance host tolerance to aggressive chemoradiotherapy for eradicating metastatic cancers.

Occurrence and regression of BK polyomavirus associated carcinoma: a clinical and next-generation sequencing study.

Low-level BK polyomavirus (BKPyV) shedding is seen in at least 10% of seropositive immunocompetent adults. Moreover, BKPyV infection is highly prevalent amongst immunocompromised populations, yet little is known on its relationship with malignancy. We studied a female patient with BKPyV-associated and donor-derived de novo high-grade sarcomatoid urothelial carcinoma developed 8 years after kidney transplantation from a male donor. Through whole-genome sequencing, we discovered integration of genotype IV BKPyV genome into the non-coding RNA (ncRNA) intronic region of human chromosome 18. The two breakpoints in the virus genome were located at the non-coding control region (NCCR) and large T antigen (TAg) coding region, respectively. Nevertheless, the TAg was overexpressed. We, therefore, inferred that the BKPyV was clonally integrated into the human genome in the form of concatemers, facilitating the expression of the TAg. The patient presented with multiorgan metastases, which were reduced in size and number throughout the body after removal of the graft and cessation of immunosuppressants. The few remaining lesions located in the liver were identified, through biopsy to be necrotic tumor tissue with TAg detected; additionally, genomic sequencing of the liver mass found Y chromosome. In conclusion, we propose that integration of the BKPyV genome is closely related to oncogenesis in this patient; while oncogenesis occurred when host immunity was impaired, recovery of the patient's native immunity effectively curbed viral replication and eliminated the metastatic lesions.

Pharmacokinetic and metabolomic analyses of the neuroprotective effects of salvianolic acid A in a rat ischemic stroke model.

Salvianolic acid A (SAA), a water-soluble phenolic acid isolated from the root of Dan Shen, displays distinct antioxidant activity and effectiveness in protection against cerebral ischemia/reperfusion (I/R) damage. However, whether SAA can enter the central nervous system and exert its protective effects by directly targeting brain tissue remains unclear. In this study, we evaluated the cerebral protection of SAA in rats subjected to transient middle cerebral artery occlusion (tMCAO) followed by reperfusion. The rats were treated with SAA (5, 10 mg/kg, iv) when the reperfusion was performed. SAA administration significantly decreased cerebral infarct area and the brain water content, attenuated the neurological deficit and pathology, and enhanced the anti-inflammatory and antioxidant capacity in tMCAO rats. The concentration of SAA in the plasma and brain was detected using LC-MS/MS. A pharmacokinetic study revealed that the circulatory system exposure to SAA was equivalent in the sham controls and I/R rats, but the brain exposure to SAA was significantly higher in the I/R rats than in the sham controls (fold change of 9.17), suggesting that the enhanced exposure to SAA contributed to its cerebral protective effect. Using a GC/MS-based metabolomic platform, metabolites in the serum and brain tissue were extracted and profiled. According to the metabolomic pattern of the tissue data, SAA administration significantly modulated the I/R-caused perturbation of metabolism in the brain to a greater extent than that in the serum, demonstrating that SAA worked at the brain tissue level rather than the whole circulation system. In conclusion, a larger amount of SAA enters the central nervous system in ischemia/reperfusion rats to facilitate its protective and regulatory effects on the perturbed metabolism.

In vitro assessment of the glucose-lowering effects of berberrubine-9-O-ß-D-glucuronide, an active metabolite of berberrubine.

Berberrubine (BRB) is the primary metabolite of berberine (BBR) that has shown a stronger glucose-lowering effect than BBR in vivo. On the other hand, BRB is quickly and extensively metabolized into berberrubine-9-O-ß-D-glucuronide (BRBG) in rats after oral administration. In this study we compared the pharmacokinetic properties of BRB and BRBG in rats, and explored the mechanisms underlying their glucose-lowering activities. C57BL/6 mice with HFD-induced hyperglycemia were administered BRB (50 mg·kg-1·d-1, ig) for 6 weeks, which caused greater reduction in the plasma glucose levels than those caused by BBR (120 mg·kg-1·d-1) or BRB (25 mg·kg-1·d-1). In addition, BRB dose-dependently decreased the activity of α-glucosidase in gut of the mice. After oral administration of BRB in rats, the exposures of BRBG in plasma at 3 different dosages (10, 40, 80 mg/kg) and in urine at different time intervals (0-4, 4-10, 10-24 h) were dramatically greater than those of BRB. In order to determine the effectiveness of BRBG in reducing glucose levels, we prepared BRBG from the urine pool of rats, and identified and confirmed it through LC-MS-IT-TOF and NMR spectra. In human normal liver cell line L-O2 in vitro, treatment with BRB or BRBG (5, 20, 50 µmol/L) increased glucose consumption, enhanced glycogenesis, stimulated the uptake of the glucose analog 2-NBDG, and modulated the mRNA levels of glucose-6-phosphatase and hexokinase. However, both BBR and BRB improved 2-NBDG uptake in insulin-resistant L-O2 cells, while BRBG has no effect. In conclusion, BRB exerts a stronger glucose-lowering effect than BBR in HFD-induced hyperglycemia mice. Although BRB significantly stimulated the insulin sensitivity and glycolysis in vitro, BRBG may have a greater contribution to the glucose-lowering effect because it has much greater system exposure than BRB after oral administration of BRB. The results suggest that BRBG is a potential agent for reducing glucose levels.

Long-Term Exposure to Air Pollution and Increased Risk of Membranous Nephropathy in China.

The effect of air pollution on the changing pattern of glomerulopathy has not been studied. We estimated the profile of and temporal change in glomerular diseases in an 11-year renal biopsy series including 71,151 native biopsies at 938 hospitals spanning 282 cities in China from 2004 to 2014, and examined the association of long-term exposure to fine particulate matter of <2.5 µm (PM2.5) with glomerulopathy. After age and region standardization, we identified IgA nephropathy as the leading type of glomerulopathy, with a frequency of 28.1%, followed by membranous nephropathy (MN), with a frequency of 23.4%. Notably, the adjusted odds for MN increased 13% annually over the 11-year study period, whereas the proportions of other major glomerulopathies remained stable. During the study period, 3-year average PM2.5 exposure varied among the 282 cities, ranging from 6 to 114 µg/m3 (mean, 52.6 µg/m3). Each 10 µg/m3 increase in PM2.5 concentration associated with 14% higher odds for MN (odds ratio, 1.14; 95% confidence interval, 1.10 to 1.18) in regions with PM2.5 concentration >70 µg/m3 We also found that higher 3-year average air quality index was associated with increased risk of MN. In conclusion, in this large renal biopsy series, the frequency of MN increased over the study period, and long-term exposure to high levels of PM2.5 was associated with an increased risk of MN.

Computed Tomographic Angiography-Based Characterization of Source Blood Vessels for Nipple-Areola Complex Perfusion in Hypertrophic Breasts.

BACKGROUND: Current knowledge about the blood supply of the nipple-areola complex (NAC) has largely been derived from studies on cadavers or persons with breasts of normal size. The aim of this study was to identify and classify the NAC blood supply by computed tomographic angiography (CTA) examination in female volunteers with breast hypertrophy. METHODS: CTA examination was performed on hypertrophic breasts of 23 female subjects. The main blood supplies were revealed through image data analyses. The dominant blood supply of the NAC and its vascular sources were identified and sorted. The detectable diameter threshold of blood vessels was set beyond 1.0 mm. RESULTS: A total of 61 dominant blood vessels were identified. The source arteries were traced as the internal thoracic artery (ITA, 50.8%), lateral thoracic artery (LTA, 27.8%), thoracoacromial artery (TA, 14.8%), brachial artery (BA, 3.3%), and axillary artery (AA, 3.3%), and the corresponding reproducibility of these source vessels was 31, 37, 9, 4.3, and 4.3%, in all breasts. The intercostal artery (IA) was not identified as a dominant NAC supplying vessel in any CTA scan image. Twenty-six breasts had only one dominant artery, whereas 17 breasts showed multiple dominant blood supplies. Three breasts showed no dominant blood vessels of the NAC, with diameters greater than the detectable threshold of 1.0 mm, and 52.2% of the breasts demonstrated anatomically symmetrical patterns of blood supply for the NAC. CONCLUSIONS: The ITA, LTA, and TA are likely to be the main vessel sources, whereas the IA is unlikely to be the dominant vessel for NAC perfusion, on the basis of the studied breasts. An asymmetrical pattern of bilateral breast blood supply was demonstrated in a considerable portion of the females with breast hypertrophy in this study. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Mst1/Mst2 regulate development and function of regulatory T cells through modulation of Foxo1/Foxo3 stability in autoimmune disease.

Foxp3 expression and regulatory T cell (Treg) development are critical for maintaining dominant tolerance and preventing autoimmune diseases. Human MST1 deficiency causes a novel primary immunodeficiency syndrome accompanied by autoimmune manifestations. However, the mechanism by which Mst1 controls immune regulation is unknown. In this article, we report that Mst1 regulates Foxp3 expression and Treg development/function and inhibits autoimmunity through modulating Foxo1 and Foxo3 (Foxo1/3) stability. We have found that Mst1 deficiency impairs Foxp3 expression and Treg development and function in mice. Mechanistic studies reveal that Mst1 enhances Foxo1/3 stability directly by phosphorylating Foxo1/3 and indirectly by attenuating TCR-induced Akt activation in peripheral T cells. Our studies have also shown that Mst1 deficiency does not affect Foxo1/3 cellular localization in CD4 T cells. In addition, we show that Mst1(-/-) mice are prone to autoimmune disease, and mutant phenotypes, such as overactivation of naive T cells, splenomegaly, and autoimmune pathological changes, are suppressed in Mst1(-/-) bone marrow chimera by cotransplanted wt Tregs. Finally, we demonstrate that Mst1 and Mst2 play a partially redundant role in Treg development and autoimmunity. Our findings not only identify Mst kinases as the long-searched-for factors that simultaneously activate Foxo1/3 and inhibit TCR-stimulated Akt downstream of TCR signaling to promote Foxp3 expression and Treg development, but also shed new light on understanding and designing better therapeutic strategies for MST1 deficiency-mediated human immunodeficiency syndrome.

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-ß1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.

VEGF-A inhibition ameliorates podocyte apoptosis via repression of activating protein 1 in diabetes.

BACKGROUND/AIMS: Vascular endothelial growth factor-A (VEGF-A) upregulation and podocyte apoptosis have been documented in diabetes. This study was designed to investigate whether inhibiting VEGF-A could ameliorate podocyte apoptosis in diabetes and the underlying mechanisms. METHODS: In vitro, small interfering RNAs (siRNAs) of VEGF-A and activator protein 1 (AP-1, c-fos and c-jun), bevacizumab (VEGF-A inhibitor) and SP600125 (AP-1 inhibitor) were added to high glucose (30 mM) induced podocytes. Luciferase reporter assay was used to investigate whether AP-1 was a direct target of VEGF-A. In vivo, bevacizumab and SP600125 were administered to 12-week-old streptozotocin-induced male Sprague Dawley rats. The level of VEGF-A, c-fos, c-jun and bcl-2 were examined using immunostaining and Western blot analysis. Podocyte apoptosis was detected using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay, electron microscopy and flow cytometry. RESULTS: Silencing VEGF-A or AP-1 upregulated bcl-2 and ameliorated podocyte apoptosis. Silencing VEGF-A decreased the level of c-fos and c-jun and bevacizumab and SP600125 treatment attenuated podocyte apoptosis. Luciferase reporter activity of VEGF-A-3'-UTR constructs was significantly provoked when stimulated with TGF-ß1. In diabetic rat kidneys, VEGF-A co-localized with bcl-2 in podocytes. With bevacizumab and SP600125 treatment, the level of VEGF-A and AP-1 decreased while bcl-2 increased. Podocyte apoptotic rate was reduced with condensed podocyte nuclei less frequently observed. The urine albumin excretion rate (UAER) and albumin/creatinine were improved. CONCLUSION: This study demonstrates VEGF-A inhibition ameliorates podocyte apoptosis by regulating AP-1 and bcl-2 signaling. AP-1 is a direct target of VEGF-A and a novel player in podocyte apoptosis.