Background: Fatigue persisting post-treatment is commonly reported by thyroid cancer (TC) survivors. Methods: We conducted a systematic review of published English language randomized controlled trials (RCTs) on interventions for management of persistent post-treatment fatigue in TC. Our review excluded studies on short-term interventions used in preparation for radioactive iodine diagnostic scans or treatment. An electronic search was executed in 6 databases and supplemented by a hand search. Two reviewers independently reviewed all citations from the electronic search and relevant full-text papers. Two abstractors independently critically appraised included papers and abstracted the data. The data were qualitatively summarized. Results: We reviewed a total of 1086 unique citations and 25 full-text papers. Four papers summarizing the results of 3 RCTs were included. The interventions included: combination tri-iodothyronine with levothyroxine (L-T4) therapy compared to L-T4 alone (1 RCT), reduction in degree of thyrotropin (TSH) suppression using L-T4 compared to maintenance of TSH suppression (1 RCT), and supervised exercise compared to inactivity (2 RCTs examining different fatigue outcomes in same population). Trial duration ranged from 10 weeks to 6 months. All trials had limitations, and the number of TC survivors included in respective RCTs ranged from 15 to 36. Hormonal treatment RCTs had mixed fatigue outcome results within respective trials, however, multiple measures suggesting improvement in fatigue were reported following the exercise intervention. Conclusions: There is paucity of RCTs to guide evidence-based management of persistent-post-treatment fatigue in TC survivors. RCTs of interventions for prevention or treatment of fatigue in TC survivors are needed.
Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K(+) reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K(+) secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)-components of an E3 ubiquitin ligase complex that targets WNKs for degradation-cause constitutively increased renal salt reabsorption and impaired K(+) secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3(S433) phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K(+) secretion in the setting of volume depletion.
The renal phenotype induced by loss-of-function mutations of inwardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which Kir.4.1 mutations cause the tubulopathy is not completely understood. Here we demonstrate that Kcnj10 is a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and determines the expression of the apical Na-Cl cotransporter (NCC) in the DCT. Immunostaining demonstrated Kcnj10 and Kcnj16 were expressed in the basolateral membrane of DCT, and patch-clamp studies detected a 40-pS K channel in the basolateral membrane of the DCT1 of p8/p10 wild-type Kcnj10(+/+) mice (WT). This 40-pS K channel is absent in homozygous Kcnj10(-/-) (knockout) mice. The disruption of Kcnj10 almost completely eliminated the basolateral K conductance and decreased the negativity of the cell membrane potential in DCT1. Moreover, the lack of Kcnj10 decreased the basolateral Cl conductance, inhibited the expression of Ste20-related proline-alanine-rich kinase and diminished the apical NCC expression in DCT. We conclude that Kcnj10 plays a dominant role in determining the basolateral K conductance and membrane potential of DCT1 and that the basolateral K channel activity in the DCT determines the apical NCC expression possibly through a Ste20-related proline-alanine-rich kinase-dependent mechanism.
Phenylethanolamine A (PEA) is a phenethanolamine member of the family of ?-adrenergic agonists compounds illegally used as feed additives for growth promotion. In this study, PEA was covalently linked to carrier protein cationized bovine serum albumin (cBSA) and egg albumin (OVA). A monoclonal antibody specific for PEA was produced and characteristics of monoclonal antibody (McAb) were studied. Based on the McAb, an enzyme-linked immunosorbent assay (ELISA) for detection of PEA was developed, which showed an IC50 value of 6.25 ng mL(-1) and a detection limit of 0.19 ng mL(-1). The average recovery of PEA from spiked samples was 103.4%, which demonstrated that the matrices of meat where PEA may be found do not interfere with the assay.
The surgical management with laparoscopic technique for renal cell carcinoma with inferior vena cava tumor thrombus (IVTT) remains challenging and technically demanding in urological oncology. We present two patients with level II IVTT that were managed with pure conventional laparoscopic radical nephrectomy and thrombectomy. Two patients were diagnosed with a renal tumor with level II IVTT from December 2011 to January 2012. They both underwent pure conventional laparoscopic radical nephrectomy with thrombectomy. During these operations, intraoperative laparoscopic ultrasonography was used to detect the thrombus and ensure complete removal. Two patients were operated through retroperitoneal approach for right renal tumor and transperitoneal approach for left renal tumor respectively. The demographics, perioperative and follow-up data were recorded for the study. Both operations were successfully performed without conversion. They both had no radiographic evidence of recurrence during follow-up. It is concluded that it is feasible to manage renal cell carcinoma with level II IVTT through pure conventional laparoscopic approach in carefully selected patients, which might expand the indication for laparoscopic surgery. The purê laparoscopic approach in the treatment of renal cell carcinoma with level II vena cava tumor thrombus is challenging and requires advanced laparoscopic skills. Multicenter prospective randomized control trials are needed to prove the benefits of this approach.
Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here, we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors and to elucidate the molecular mechanisms underlying chondrogenesis.
Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/?-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells.
China has experienced a sharply increasing rate of human brucellosis in recent years. Effective spatial monitoring of human brucellosis incidence is very important for successful implementation of control and prevention programmes. The purpose of this paper is to apply exploratory spatial data analysis (ESDA) methods and the empirical Bayes (EB) smoothing technique to monitor county-level incidence rates for human brucellosis in mainland China from 2004 to 2010 by examining spatial patterns.
Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPAR?2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.
Laparoendoscopic single-site (LESS) surgery through the retroperitoneal approach has been seldom reported. We aimed to evaluate the outcomes of LESS and conventional laparoscopic surgery via the retroperitoneal approach in the management of large, impacted ureteral calculi.
Osteosarcoma (OS) is the most common primary malignancy of bone and is usually associated with poor prognosis due to its high incidence of metastasis and chemoresistance. Molecular pathogenesis of OS is poorly understood. We previously showed that OS cells are refractory to BMP9-induced osteogenesis and respond favorably to proliferation and tumor growth. Here we investigate if Notch signaling mediates the BMP9-promoted cell proliferation and tumor growth of human osteosarcoma (OS). We find that the expression of Notch1, Notch2, Notch3, DLL1, JAG1 and JAG2 is readily detected in most of the tested OS cell lines. BMP9-promoted OS cell proliferation, migration, and cell cycle S/G2 progression are effectively inhibited by a dominant-negative mutant of Notch1 (dnNotch1) or the ?-secretase inhibitor Compound E (ComE). Furthermore, BMP9-promoted tumor growth and osteolytic lesions in vivo are significantly inhibited by dnNotch1. BMP9 up-regulates the expression of Notch1, Notch3, DLL1, and JAG1 in OS cells. Accordingly, BMP9 stimulation induces a nuclear accumulation of NICD, which is blocked by ComE. Our results demonstrate that BMP9-promoted OS proliferation and tumor growth is at least in part mediated by Notch signaling, suggesting that osteogenic BMPs may function as upstream regulators of Notch signaling in OS tumorigenesis. Thus, pharmacologic intervention of Notch signaling may be explored as a new therapeutic strategy for human OS tumors.
Metal-organic frameworks (MOFs) have been explored for analytical applications because of their outstanding properties such as high surface areas, flexibility and specific structure features, especially for chromatography application in recent years. In this work, a chiral MOF Ni(D-cam)(H2O)2 with unusual integration of molecular chirality, absolute helicity, and 3-D intrinsic chiral net was chosen as stationary phase to prepare Ni(D-cam)(H2O)2-coated open tubular columns for high-resolution gas chromatographic (GC) separation. Two fused-silica open tubular columns with different inner diameters and lengths, including column A (30 m × 250 µm i.d.) and column B (2 m × 75 µm i.d.), were prepared via a dynamic coating method. The chromatographic properties of the two columns were investigated using n-dodecane as the analyte at 120 °C. The number of theoretical plates (plates/m) of the two metal-organic framework (MOF) columns was 1300 and 2750, respectively. The racemates, isomer and linear alkanes mixture were used as analytes for evaluating the separation properties of Ni(D-cam)(H2O)2-coated open tubular columns. The results showed that the columns offered good separations of isomer and linear alkanes mixture, especially racemates.
Ecosystem light use efficiency (LUE) is a key factor of production models for gross primary production (GPP) predictions. Previous studies revealed that ecosystem LUE could be significantly enhanced by an increase on diffuse radiation. Under large spatial heterogeneity and increasing annual diffuse radiation in China, eddy covariance flux data at 6 sites across different ecosystems from 2003 to 2007 were used to investigate the impacts of diffuse radiation indicated by the cloudiness index (CI) on ecosystem LUE in grassland and forest ecosystems. Our results showed that the ecosystem LUE at the six sites was significantly correlated with the cloudiness variation (0.24?R2?0.85), especially at the Changbaishan temperate forest ecosystem (R2?=?0.85). Meanwhile, the CI values appeared more frequently between 0.8 and 1.0 in two subtropical forest ecosystems (Qianyanzhou and Dinghushan) and were much larger than those in temperate ecosystems. Besides, cloudiness thresholds which were favorable for enhancing ecosystem carbon sequestration existed at the three forest sites, respectively. Our research confirmed that the ecosystem LUE at the six sites in China was positively responsive to the diffuse radiation, and the cloudiness index could be used as an environmental regulator for LUE modeling in regional GPP prediction.
Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs' basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.
Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 ?g/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 ?g/ml and 2 ?g/ml, respectively. FACS analysis indicates that Polybrene (at 4 ?g/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 ?g/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 ?g/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.
Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults. Molecular mechanisms underlying the pathogenesis of OS remain to be fully understood. Several members of the E-F hand calcium-binding S100 protein family are differentially expressed in human cancers. We previously showed that S100A6 is highly expressed in OS tumors. In this study, we investigated the role of S100A4 in regulating OS proliferation and osteogenic differentiation.
Successful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell-based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically-used scaffold materials, type I collagen sponge, hydroxyapatite-tricalcium phosphate (HA-TCP) and demineralized bone matrix (DBM), using BMP9-expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus-mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9-transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9-expressing C2C12 cells with collagen sponge or HA-TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA-TCP scaffold carriers may provide more cell-friendly environment to support the survival, propagation, and ultimately differentiation of BMP9-expressing progenitor cells. This line of investigation should provide important experimental evidence for further pre-clinical studies in BMP9-mediated cell based approach to bone tissue engineering.
Recently, interest in the utilization of corncob residue (CCR, with high lignin of 45.1%) as a feedstock for bioethanol has been growing. Surfactants have been one of the most popular additives intended to prevent the inhibitory effect of lignin on cellulolytic enzymes, thereby improving hydrolysis. In this study, the effects of biosurfactant tea saponin (TS) on the enzymatic hydrolysis of CCR and the bonding behavior of cellulolytic enzymes to the substrate were investigated. The surface tension in the supernatant was also detected to obtaininformation about the characteristics and stability of TS.
Nuclear receptors are transcription factors that regulate diverse cellular processes. In canonical activation, ligand availability is sufficient to produce receptor binding, entraining downstream signaling. The mineralocorticoid receptor (MR) is normally activated by aldosterone, which is produced in both volume depletion and hyperkalemia, states that require different homeostatic responses. We report phosphorylation at S843 in the MR ligand-binding domain that prevents ligand binding and activation. In kidney, MR(S843-P) is found exclusively in intercalated cells of the distal nephron. In volume depletion, angiotensin II and WNK4 signaling decrease MR(S843-P) levels, whereas hyperkalemia increases MR(S843-P). Dephosphorylation of MR(S843-P) results in aldosterone-dependent increases of the intercalated cell apical proton pump and Cl(-)/HCO3(-) exchangers, increasing Cl(-) reabsorption and promoting increased plasma volume while inhibiting K(+) secretion. These findings reveal a mechanism regulating nuclear hormone receptor activity and implicate selective MR activation in intercalated cells in the distinct adaptive responses to volume depletion and hyperkalemia.
To perform a comprehensive evaluation and comparison of tear function in diabetic and non-diabetic patients. Research related to tear function in diabetic and non-diabetic patients was gathered using PubMed, EBSCO, OVID. Two reviewers independently conducted the literature search. The quality assessment and the data extraction were performed in accordance with exclusion criteria and cross-checking. RevMan 5.1.7 software was used for the meta-analysis. The tear film break-up time was studied in eight articles with a total of 1449 samples. Through a random-effects model analysis, the combined weighted mean difference (WMD) was -4.44 [-5.87, -3.01]. The time in diabetic patients was shorter than that in the non-diabetic group (p?0.00001). The basal tear secretion test was studied in seven articles with a total of 949 samples. The combined WMD was -3.96 [-5.70, -2.23], and the difference between the diabetic group and control group was statistically significant (p?0.00001). The total tear secretion test was studied in five articles with a total of 921 samples. The combined WMD was -3.96 [-7.43, -0.50]. The difference between the diabetic and control groups was statistically significant (p?=?0.03). The corneal sensitivity was compared in eight studies with a total of 976 samples. Through a random-effects model analysis, the standardized mean difference (SMD) was -5.14 [-6.99, -3.29]. The corneal sensitivity was lower in diabetic patients than the control group (p?0.00001). Our study suggests that the tear functions are worse in diabetic patients compared with the control group. Moreover, patients with PDR are more predisposed to impaired tear functions.
Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K(+) secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complexs ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K(+) channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3-RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4.
Osteogenic differentiation from mesenchymal progenitor cells (MPCs) are initiated and regulated by a cascade of signaling events. Either Wnt/?-catenin or estrogen signaling pathway has been shown to play an important role in regulating skeletal development and maintaining adult tissue homeostasis. Here, we investigate the potential crosstalk and synergy of these two signaling pathways in regulating osteogenic differentiation of MPCs. We find that the activation of estrogen receptor (ER) signaling by estradiol (E2) or exogenously expressed ER? in MPCs synergistically enhances Wnt3A-induced early and late osteogenic markers, as well as matrix mineralization. The E2 or ER?-mediated synergy can be effectively blocked by ER? antagonist tamoxifen. E2 stimulation can enhance endochondral ossification of Wnt3A-transduced mouse fetal limb explants. Furthermore, exogenously expressed ER? significantly enhances the maturity and mineralization of Wnt3A-induced subcutaneous and intramuscular ectopic bone formation. Mechanistically, we demonstrate that E2 does not exert any detectable effect on ?-catenin/Tcf reporter activity. However, ER? expression is up-regulated within the first 48h in AdWnt3A-transduced MPCs, whereas ER? expression is significantly inhibited within 24h. Moreover, the key enzyme for the biosynthesis of estrogens aromatase is modulated by Wnt3A in a biphasic manner, up-regulated at 24h but reduced after 48h. Our results demonstrate that, while ER signaling acts synergistically with Wnt3A in promoting osteogenic differentiation, Wnt3A may crosstalk with ER signaling by up-regulating ER? expression and down-regulating ER? expression in MPCs. Thus, the signaling crosstalk and synergy between these two pathways should be further explored as a potential therapeutic approach to combating bone and skeletal disorders, such as fracture healing and osteoporosis.
Aberrant activation of ?-catenin signaling plays an important role in human tumorigenesis. However, molecular mechanisms behind the ?-catenin signaling deregulation are mostly unknown because genetic alterations in this pathway only account for a small fraction of tumors. Here, we investigator if other major pathways can regulate ?-catenin signaling activity. By employing a panel of chemical activators and/or inhibitors of several cellular signaling pathways, we assess these modulators effects on luciferase reporter driven by ?-catenin/TCF4-responsive elements. We find that lithium-stimulated ?-catenin activity is synergistically enhanced by protein kinase C activator PMA. However, ?-catenin-regulated transcriptional (CRT) activity is significantly inhibited by casein kinase II inhibitor DRB, MEK inhibitor PD98059, G-proteins and their receptor uncoupling agent suramin, protein tyrosine kinase inhibitor genistein, and PI-3 kinase inhibitor wortmannin, suggesting that these cellular pathways may participate in regulating ?-catenin signaling. Interestingly, the Ca(++)/calmodulin kinase II inhibitor HDBA is shown to activate ?-catenin activity at low doses. Furthermore, Wnt3A-stimulated and constitutively activated CRT activities, as well as the intracellular accumulation of ?-catenin protein in human colon cancer cells, are effectively suppressed by PD98059, genistein, and wortmannin. We further demonstrate that EGF can activate TCF4/?-catenin activity and induce the tyrosine phosphorylation of ?-catenin protein. Thus, our results should provide important insights into the molecular mechanisms underlying Wnt/?-catenin activation. This knowledge should facilitate our efforts to develop efficacious and novel therapeutics by targeting these pathways.
Glucocorticoid-mediated hypertension is incompletely understood. Recent studies have suggested the primary mechanism of this form of hypertension may be through the effects of glucocorticoids on vascular tissues and not to excess sodium and water re-absorption as traditionally believed.
WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, was reported to be downregulated in gastric cancer and other tumors. However, the mechanism by which WWOX is inactivated remains unclear. In our study, methylation status of WWOX was determined by MSP and sequencing. Our results showed that WWOX hypermethylation was frequently detected in gastric cancer, and also significantly correlated with Helicobacter pylori (H. pylori) infection. Promoter methylation of WWOX was induced in BCG823 and AGS cells co-cultured with H. pylori. Finally, we found that expression of DNMT1 and DNMT3A were enhanced when cells were co-cultured with H. pylori. Our study indicated that H. pylori infection promoted methylation of WWOX gene in gastric cancer.
Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays a critical role in cell motility. Movement and retraction of podocyte foot processes, which accompany podocyte injury, suggest focal adhesion disassembly. To understand better the mechanisms by which podocyte foot process effacement leads to proteinuria and kidney failure, we studied the function of FAK in podocytes. In murine models, glomerular injury led to activation of podocyte FAK, followed by proteinuria and foot process effacement. Both podocyte-specific deletion of FAK and pharmacologic inactivation of FAK abrogated the proteinuria and foot process effacement induced by glomerular injury. In vitro, podocytes isolated from conditional FAK knockout mice demonstrated reduced spreading and migration; pharmacologic inactivation of FAK had similar effects on wild-type podocytes. In conclusion, FAK activation regulates podocyte foot process effacement, suggesting that pharmacologic inhibition of this signaling cascade may have therapeutic potential in the setting of glomerular injury.
Mercury ions (Hg(2+)) are one of the most dangerous pollutants. Even at low concentration, it causes serious environmental and health problems. Current methods for the detection of Hg(2+) in environmental samples are tedious and time consuming because they require sophisticated instrumentation and complicated sample pre-treatment processes. In this work, a novel probe with high selectivity towards Hg(2+) was synthesized and a one step competitive immunochromatographic assay based on the probe for the detection of Hg(2+) was developed and applied for water samples. The detection conjugate was immobilized on one end of the nitrocellulose membrane (detection line) and anti-BSA polyclonal antibody was immobilized on the other end of the membrane (control line). Hg(2+) in samples competed with the probe to bind with immobilized detection conjugate. The visual detection limit of Hg(2+) in spiked water samples was found to be about 1 ppb. The qualitative assay can be performed within 15 min. The advantages of the technique are rapidity, low cost and without the need of any equipment and complicated sample preparation.
Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.
Inactivating mutations of PHEX cause X-linked hypophosphatemia and result in increased circulating fibroblast growth factor 23 (FGF23). FGF23 action is dependent upon Klotho, which converts FGF receptor 1 into an FGF23-specific receptor. Disruption of Klotho results in a complex bone phenotype and hyperphosphatemia, the converse phenotype of X-linked hypophosphatemia. We examined effects of disrupting both Klotho and PHEX by creating a double-knockout (Klotho/HYP) mouse. The combined disruption corrected the hypophosphatemia in HYP mice, indicating that Klotho is epistatic to PHEX. FGF23 levels remained elevated in all groups except wild-type, indicating that Klotho is necessary for FGF23-dependent phosphaturic activity. 1,25-Dihydroxyvitamin D levels, reduced in HYP mice, were comparably elevated in Klotho and Klotho/HYP mice, demonstrating that Klotho is necessary for FGF23s effect on vitamin D metabolism. Serum PTH levels were reduced in both Klotho and Klotho/HYP mice. Moreover, the Klotho null phenotype persisted in Klotho/HYP, maintaining the runty phenotype and decreased life span of Klotho null mice. Notably, microcomputed tomography analysis demonstrated greater trabecular bone volume fraction in Klotho/HYP mice than that in all other groups (Klotho/HYP, 56.2 +/- 6.3%; Klotho, 32.5 +/- 10.3%; HYP, 8.6 +/- 7.7%; and wild type, 21.4 +/- 3.4%; P < 0.004). Histomorphometric analysis confirmed the markedly increased trabecular bone density in Klotho/HYP mice and the well-established increase in osteoid volume in HYP mice. These observations suggest that with addition of Klotho loss of function, the overabundant osteoid typically produced in HYP mice (but fails to mineralize) is produced and mineralized in the double knockout, resulting in markedly enhanced trabecular bone density.
Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3s intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.
Glucocorticoids (GCs) exert profound influences on many physiologic functions by virtue of their diverse roles in growth, development, and maintenance of homeostasis. We previously created a novel gain of function in the human glucocorticoid receptor (hGR), hGRM604L, which is active at GC concentrations 5-10-fold lower than wild-type GR. To gain a greater insight into GC physiology in vivo, we inserted this mutant GR (GRM610L in mice) into mice via homologous recombination. Mice expressing the allele are phenotypically normal with respect to GC function. However, corticosterone levels, ACTH levels, and adrenocortical size are markedly reduced, suggesting they are phenotypically normal because the mutant GR alters the basal regulation of the hypothalamic-pituitary-adrenal axis. We demonstrate via physiologic and immunologic studies that GRM610L mice have increased sensitivity to GCs in vivo. Sensitivity to the actions of endogenous GCs may be an important factor underlying the development of many human diseases including hypertension, obesity, and diabetes. Our model may provide a new and powerful tool for the study of GC physiological and pathological processes in vivo.
Renal transplant recipients have a high risk of developing multiple and invasive urothelial tumors because of long-term immunosuppression and infections with oncogenic viruses in China. However, treatment of renal transplant recipients who developed invasive bladder tumor is challenging. We aimed to evaluate the efficacy and safety of orthotopic ileal neobladder reconstruction following radical cystectomy in renal transplant recipients. Orthotopic ileal neobladder reconstruction and preservation of the transplanted kidney were performed in two patients after one and 36 months of transplantation, respectively. One recipient was lacking a bladder because of prior cystectomy before the transplantation, and the other developed multiple and invasive bladder cancer after the transplantation. During the 14-month and seven-yr follow-up postoperation, no serious complications occurred except slight hydronephrosis in one patient. No rejection and graft dysfunction occurred in both patients with reduced dosage of immunosuppressants, and serum creatinine as a marker of renal function remained stable. Urinary continence was satisfactory during the day and night with voluntary voiding. Our experience showed that radical cystectomy and orthotopic ileal neobladder reconstruction in transplant patients with stable renal function is a safe and effective way to provide better quality of life, satisfactory urinary diversion and preservation of renal function simultaneously.
Ureteric bud (UB) branching during kidney development determines the final number of nephrons. Although hepatocyte growth factor and its receptor Met have been shown to stimulate branching morphogenesis in explanted embryonic kidneys, loss of Met expression is lethal during early embryogenesis without obvious kidney abnormalities. Met(fl/fl);HoxB7-Cre mice, which lack Met expression selectively in the UB, were generated and found to have a reduction in final nephron number. These mice have increased Egf receptor expression in both the embryonic and adult kidney, and exogenous Egf can partially rescue the branching defect seen in kidney explants. Met(fl/fl);HoxB7-Cre;wa-2/wa-2 mice, which lack normal Egfr and Met signaling, exhibit small kidneys with a marked decrease in UB branching at E14.5 as well as a reduction in final glomerular number. These mice developed progressive interstitial fibrosis surrounding collecting ducts with kidney failure and death by 3-4 weeks of age. Thus, in support of previous in vitro findings, Met and the Egf receptor can act cooperatively to regulate UB branching and mediate maintenance of the normal adult collecting duct.
Nanog plays a key role in stem cell self-renewal and pluripotency differentiation in embryonic stem cells ( ESCs). Recently, some studies reported that abnormal expression of Nanog could be detected in several tumors, indicating that Nanog might be related to tumor development. However, studies on the correlation between Nanog expression and esophageal cancer are sparse.
The nitrogen (N) deposition fluxes were investigated in eight typical forest ecosystems along the North-South Transect of Eastern China (NSTEC; based on the ChinaFLUX network) by ion-exchange resin (IER) columns from May 2008 to April 2009. Our results demonstrated that the method of IER columns was both labor cost saving and reliable for measuring dissolved inorganic nitrogen (DIN) deposition at the remote forest stations. The deposition of DIN in the throughfall ranged from 1.3 to 29.5 kg N ha(-1) a(-1), increasing from north to south along NSTEC. The relatively high average ratio of ammonium to nitrate in deposition (1.83) indicated that the N deposition along the NSTEC in China mostly originated in farming and animal husbandry rather than in industry and vehicle activities. For seasonal variability, the DIN deposition showed a single peak in the growing season in the northern part of NSTEC, while, in the southern part, it exhibited double-peaks in the early spring and the mid-summer, respectively. On the annual scale, the DIN deposition variations of the eight sites could be mainly explained by precipitation and the distances from forest stations to provincial capital cities.
The acaricidal activity of trans-cinnamaldehyde was evaluated in vitro on Psoroptes cuniculi. In this study, different concentrations of trans-cinnamaldehyde were tested, and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R®) controls. The morphological changes in P. cuniculi treated with trans-cinnamaldehyde were examined with light microscopy. By the analysis of variance one-way test, up to 8 ?g/ml of trans-cinnamaldehyde gave highly significant (P?0.01) percentages of mite mortality compared with the untreated controls, but only up to 256 ?g/ml, it showed the same efficacy of Acacerulen R®. At the same time, a bioassay was conducted by exposing mites to varying doses of trans-cinnamaldehyde in vitro cultures. The resulting data were analyzed by using a time-dose-mortality modeling technique, yielding the parameters for time and dose effects of P. cuniculi. The ? value was 2.01, indicating that trans-cinnamaldehyde had a good activity to kill P. cuniculi adults. Based on the time-dose-mortality relationships fitted and the virulence indices estimated, trans-cinnamaldehyde is a promising microbial agent for mites control.
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