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1.  Protein tyrosine phosphatase SHP2 regulates TGF-β1 production in airway epithelia and asthmatic airway remodeling in mice 
Allergy  2012;67(12):1547-1556.
Background
Transforming growth factor (TGF)-β1 produced in airway epithelia has been suggested as a contributor to the airway remodeling observed in asthma patients. The protein tyrosine phosphatase SHP2 is a demonstrable modulator of TGF-β1 production and thus a potential regulator of airway remodeling.
Objectives
To define the signal event by which SHP2 regulates asthmatic responses in airway epithelial cells by using a mouse model of experimental OVA-induced airway remodeling.
Methods
The airways of Shp2flox/flox mice were infected with recombinant adenovirus vectors expressing a Cre recombinase–green fluorescence protein (GFP) fusion protein as part of allergen provocation studies using mice sensitized with ovalbumin (OVA) and repeatedly challenged with OVA. Several endpoint pathologies were assessed, including airway hyper-responsiveness (AHR), lung inflammatory score, peribronchial collagen deposition, and α-smooth muscle actin (SMA) hyperplasia. In vitro studies using airway epithelial cells (BEAS-2B) were used to investigate the role of SHP2 in the regulation of pulmonary remodeling events, including the expression of collagen, α-SMA, and TGF-β1.
Results
Chronic OVA challenges in wild-type mice resulted in airway remodeling and lung dysfunction (e.g., increased inflammatory scores, collagen deposition (fibrosis), smooth muscle hyperplasia, and a significant increase in AHR). These endpoint pathology metrics were each significantly attenuated by conditional shp2 gene knockdown in airway epithelia. In vitro studies using BEAS-2B cells also demonstrated that the level of TGF-β1 production by these cells correlated with the extent of shp2 gene expression.
Conclusions
SHP2 activities in airway epithelial cells appear to modulate TGF-β1 production and, in turn, regulate allergic airway remodeling following allergen provocation.
Clinical Implications
Our findings identify SHP2 as a previously underappreciated contributor to the airway remodeling and lung dysfunction associated with allergen challenge. As such, SHP2 represents a potentially novel therapeutic target for the treatment of asthmatics.
doi:10.1111/all.12048
PMCID: PMC3942166  PMID: 23057634
airway epithelia; asthma; mice; protein tyrosine phosphatase SHP2; remodeling
2.  STK31/TDRD8, a Germ Cell-Specific Factor, Is Dispensable for Reproduction in Mice 
PLoS ONE  2014;9(2):e89471.
Tudor domain containing (Tdrd) proteins that are expressed in germ cells are divided into two groups. One group, consisting of TDRD1, TDRKH, TDRD9 and TDRD12, function in piRNA biogenesis and retrotransposon silencing, while the other group including RNF17/TDRD4 and TDRD5-7 are required for spermiogenesis. These Tdrd proteins play distinct roles during male germ cell development. Here, we report the characterization of STK31/TDRD8 in mice. STK31 contains a tudor domain and a serine/threonine kinase domain. We find that STK31 is a cytoplasmic protein in germ cells. STK31 is expressed in embryonic gonocytes of both sexes and postnatal spermatocytes and round spermatids in males. Disruption of the tudor domain and kinase domain of STK31 respectively does not affect fertility in mice. Our data suggest that the function of STK31 may be redundant with other Tdrd proteins in germ cell development.
doi:10.1371/journal.pone.0089471
PMCID: PMC3929691  PMID: 24586802
3.  Suppression of tumor angiogenesis by targeting the protein neddylation pathway 
Cell Death & Disease  2014;5(2):e1059-.
Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin–RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.
doi:10.1038/cddis.2014.21
PMCID: PMC3944239  PMID: 24525735
neddylation; MLN4924; tumor angiogenesis; cullin–RING ligase
4.  MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2 
Ruan, Q | Wang, P | Wang, T | Qi, J | Wei, M | Wang, S | Fan, T | Johnson, D | Wan, X | Shi, W | Sun, H | Chen, Y H
Cell Death & Disease  2014;5(2):e1095-.
MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.
doi:10.1038/cddis.2014.47
PMCID: PMC3944261  PMID: 24577093
MiR-21; TNFAIP8; Tipe2; apoptosis; NF-κB
5.  Efficacy and Safety of 1 % Terbinafine Film-Forming Solution in Chinese Patients with Tinea Pedis: A Randomized, Double-Blind, Placebo-Controlled, Multicenter, Parallel-Group Study 
Clinical Drug Investigation  2014;34:223-230.
Background and objectives
Superficial fungal skin infections are treated using topical antifungals. The aim of this study was to demonstrate the efficacy of a single application of 1 % terbinafine film-forming solution (FFS) versus placebo for the treatment of tinea pedis in the Chinese population.
Methods
Six centers in China randomized 290 patients in a 1:1 ratio to receive either 1 % terbinafine FFS or FFS vehicle (placebo) once on the affected foot/feet. Efficacy assessments included microscopy and mycologic culture, and assessing clinical signs and symptoms at baseline, and at weeks 1 and 6 after the topical treatment. All adverse events were recorded.
Results
At week 6, 1 % terbinafine FFS was superior to placebo for effective treatment rate (63 vs. 8 %); clinical cure (30 vs. 6 %); mycological cure (86 vs. 12 %); negative microscopy (90 vs. 24 %); and negative mycological culture (90 vs. 27 %): all p ≤ 0.001 and clinically relevant. At week 6, 1 % terbinafine FFS was clinically superior to placebo for the absence of: erythema (69 vs. 29 %); desquamation (33 vs. 8 %); and pruritus (70 vs. 30 %): all p ≤ 0.001 and clinically relevant. At week 6, differences in the average total signs and symptoms scores were significantly lower for 1 % terbinafine FFS versus placebo (p ≤ 0.001). Both 1 % terbinafine FFS and placebo were safe and well tolerated based on adverse events and investigator and patient assessments.
Conclusions
This double-blind, randomized, multicenter study demonstrated one single topical application of 1 % terbinafine FFS was safe and effective in the treatment of tinea pedis in the Chinese population.
doi:10.1007/s40261-014-0171-8
PMCID: PMC3926983  PMID: 24477462
6.  Non-muscle myosin IIB is essential for cytokinesis during male meiotic cell divisions 
Developmental biology  2012;369(2):356-361.
Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.
doi:10.1016/j.ydbio.2012.07.011
PMCID: PMC3584343  PMID: 22820068
Spermatogenesis; Meiosis; Cytokinesis; NMIIB; MYH10; Mouse
7.  The impact of short daily hemodialysis on anemia and the quality of life in Chinese patients 
Anemia is a frequent complication in hemodialysis patients. Compared to conventional hemodialysis (CHD), short daily hemodialysis (sDHD) has been reported to be effective in many countries except China. The aim of the present study was to determine whether sDHD could improve anemia and quality of life (QOL) for Chinese outpatients with end-stage renal disease. Twenty-seven patients (16 males/11 females) were converted from CHD to sDHD. All laboratory values were measured before conversion (baseline), at 3 months after conversion (sDHD1), and at 6 months after conversion (sDHD2). The patient's QOL was evaluated at baseline and 6 months after conversion using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36). Hemoglobin concentration increased significantly from 107.4±7.9 g/L at baseline to 114.4±6.8 g/L (P<0.05) at sDHD1, and 118.3±8.4 g/L (P<0.001) at sDHD2 (Student paired t-test). However, the dose requirement for erythropoietin decreased from 6847.8±1057.3 U/week at baseline to 5869.6±1094.6 U/week (P<0.05) at sDHD2. Weekly stdKt/V increased significantly from 2.05±0.13 at baseline to 2.73±0.20 (P<0.001) at sDHD1, and 2.84±0.26 (P<0.001) at sDHD2. C-reactive protein decreased from baseline to sDHD1 and sDHD2, but without statistically significant differences. Physical and mental health survey scores increased in the 6 months following conversion to sDHD. sDHD may increase hemoglobin levels, decrease exogenous erythropoietin dose requirements, and improve QOL in Chinese hemodialysis patients compared to CHD. A possible mechanism for improvement of clinical outcomes may be optimized management of uremia associated with the higher efficiency of sDHD.
doi:10.1590/1414-431X20132832
PMCID: PMC3859336  PMID: 23970065
Short daily hemodialysis; Anemia; Erythropoietin; Quality of life; End-stage renal disease
8.  miR-375 is upregulated in acquired paclitaxel resistance in cervical cancer 
Shen, Y | Wang, P | Li, Y | Ye, F | Wang, F | Wan, X | Cheng, X | Lu, W | Xie, X
British Journal of Cancer  2013;109(1):92-99.
Background:
Chemo-resistance is one of the key causal factors in cancer death and emerging evidences suggest that microRNAs (miRNAs) have critical roles in the regulation of chemo-sensitivity in cancers. Cervical cancer is one of the most common malignancies in women and insensitive to chemotherapy clinically.
Methods:
The differentially expressed miRNAs in cervical squamous cell carcinoma tissues were screened by using a microarray platform (μParaflo Sanger miRBase release 13.0). The expression of miR-375 was determined by stem-loop RT–PCR using 23 clinical cervical cancer samples and 2 cervical cancer cell lines. We exogenously upregulated miR-375 expression in SiHa and Caski cells using a pre-miRNA lentiviral vector transfection and observed its impact on paclitaxel sensitivity using MTS. The cells that stably overexpressed miR-375 were subcutaneously injected into mice to determine tumour growth and chemo-sensitivity in vivo.
Results:
Twenty-one differentially expressed miRNAs were found by miRNA microarray between pro- and post-paclitaxel cervical cancer tissues. Of those, miR-375 showed consistent high expression levels across paclitaxel-treated cervical cells and tissues. Paclitaxel induced upregulated miR-375 expression in a clear dose-dependent manner. Forced overexpression of miR-375 in cervical cancer cells decreased paclitaxel sensitivity in vitro and in vivo.
Conclusion:
Collectively, our results suggest that miR-375 might be a therapeutic target in paclitaxel-resistant cervical cancer.
doi:10.1038/bjc.2013.308
PMCID: PMC3708577  PMID: 23778521
miR-375; paclitaxel; chemo-resistance; cervical cancer
9.  Identifying the target NSCLC patient for maintenance therapy: an analysis from a placebo-controlled, phase III trial of maintenance pemetrexed (H3E-MC-JMEN)† 
Annals of Oncology  2013;24(6):1534-1542.
Background
This was a post hoc analysis of patients with non-squamous histology from a phase III maintenance pemetrexed study in advanced non-small cell lung cancer (NSCLC).
Patients and methods
The six symptom items' [average symptom burden index (ASBI)] mean at baseline was calculated using the lung cancer symptom scale (LCSS). Low and high symptom burden (LSB, ASBI < 25; HSB, ASBI ≥ 25) and performance status (PS: 0, 1) subgroups were analyzed for treatment effect on progression-free survival (PFS) and overall survival (OS) using the Cox proportional hazard models adjusted for demographic/clinical factors.
Results
Significantly longer PFS and OS for pemetrexed versus placebo occurred in LSB patients [PFS: median 5.1 versus 2.4 months, hazard ratio (HR) 0.49, P < 0.0001; OS: median 17.5 versus 11.0 months, HR 0.63, P = 0.0012] and PS 0 patients (PFS: median 5.5 versus 1.7 months, HR 0.36, P < 0.0001; OS: median 17.7 versus 10.3 months, HR 0.54, P = 0.0019). Significantly longer PFS, but not OS, occurred in HSB patients (median 3.7 versus 2.8 months, HR 0.50, P = 0.0033) and PS 1 patients (median 4.4 versus 2.8 months, HR 0.60, P = 0.0002).
Conclusions
ASBI and PS are associated with survival for non-squamous NSCLC patients, suggesting that maintenance pemetrexed is useful for LSB or PS 0 patients following induction.
doi:10.1093/annonc/mdt123
PMCID: PMC3660084  PMID: 23559150
lung cancer symptom scale; maintenance therapy; non-squamous NSCLC; patient-reported symptoms; pemetrexed; survival
10.  64-slice CT angiography for the detection of functionally significant coronary stenoses: comparison with stress myocardial perfusion imaging 
The British Journal of Radiology  2012;85(1012):368-376.
Objective
To evaluate the accuracy of 64-slice CT angiography (CTA) compared with single photon emission CT (SPECT) myocardial perfusion imaging (MPI), which served as the reference standard, for the detection of functionally significant coronary artery disease (CAD).
Methods
141 consecutive patients (60±10 years, 101 men) were investigated with 64-slice CTA and SPECT MPI; a subset of 35 patients had additional invasive coronary angiography (ICA). The data from CTA and ICA were compared with those from MPI for both cut-offs of ≥50% and ≥70% stenosis, respectively.
Results
The sensitivity, specificity, positive and negative predictive values, and accuracy of CTA, using a cut-off of ≥50% for significant stenosis, in detecting inducible perfusion defects on MPI were 96% [95% confidence interval (CI) 88–100%], 61% (95% CI 52–70%), 37% (95% CI 23–49%), 99% (95% CI 97–100%) and 68%, respectively, in patient-based analysis and 97% (95% CI 91–100%), 86% (95% CI 83–89%), 33% (95% CI 24–42%), 100% (95% CI 99–100%) and 87%, respectively, in vessel-based analysis. Applying a cut-off of ≥70% for significant stenosis, CTA yielded the following sensitivity, specificity, positive and negative predictive values, and accuracy for the detection of inducible MPI defects: by patient, 65% (95% CI 46–84%), 95% (95% CI 91–99%), 74% (95% CI 50–92%), 92% (95% CI 87–97%) and 89%, respectively; by vessel, 58% (95% CI 42–74%), 97% (95% CI 95–99%), 62% (95% CI 45–79%), 97% (95% CI 95–99%) and 95%, respectively.
Conclusion
64-slice CTA is a reliable tool to exclude functionally significant CAD when using a cut-off of ≥50% diameter stenosis. By contrast, a cut-off of ≥70% diameter narrowing is a strong predictor of ischaemia.
doi:10.1259/bjr/59249210
PMCID: PMC3486657  PMID: 21224298
11.  Synovial chondromatosis of the temporomandibular joint: MRI findings with pathological comparison 
Dentomaxillofacial Radiology  2012;41(2):110-116.
Objectives
The aim of this retrospective study was to characterize MRI findings of synovial chondromatosis in the temporomandibular joint (TMJ) by correlation with their pathological findings.
Methods
22 patients with synovial chondromatosis in unilateral TMJ were referred for plain MRI prior to surgical management and pathological examinations. Parasagittal and coronal proton density-weighted imaging and T2 weighted imaging were performed for each case.
Results
MRI demonstrated multiple chondroid nodules and joint effusion in all patients (100%) and amorphous iso-intensity signal tissues within expanded joint space and capsule in 19 patients (86.4%). On T2 weighted imaging, signs of low signal nodules within amorphous iso-intensity signal tissues were used to determine the presence of attached cartilaginous nodules in pathology, resulting in 100% sensitivity, 60% specificity and 90.9% accuracy. Signs of low and intermediate signal nodules within joint fluids were used to detect loose cartilaginous nodules and resulted in 80% sensitivity, 42.9% specificity and 68.2% accuracy.
Conclusions
MRI of synovial chondromatosis in TMJ was characterized by multiple chondroid nodules, joint effusion and amorphous iso-intensity signal tissues within the expanded space and capsule. The attached cartilaginous nodules in pathology were better recognized than the loose ones on MRI. Plain MRI was useful for clinical diagnosis of the disorder.
doi:10.1259/dmfr/36144602
PMCID: PMC3520366  PMID: 22116129
synovial chondromatosis; tempromandibular joint; magnetic resonance imaging
12.  Recurrent keratocystic odontogenic tumours: report of 19 cases 
Dentomaxillofacial Radiology  2012;41(2):96-102.
Objectives
The aim of this study was to analyse 19 recurrent keratocystic odontogenic tumours (KCOTs).
Methods
19 patients with recurrent KCOTs were retrospectively analysed. These patients had been treated by either enucleation or a combination of enucleation and Carnoy's solution. The analyses covered major aspects of primary KCOT and/or recurrent KCOT identities, including patient profile, clinical features, histopathology, radiology, treatment and prognosis.
Results
19 (7.4%) out of 257 primary KCOT cases recurred, with an average patient age of 30.5 years (age range 18–45 years). 15 lesions were in the mandible and the remaining 4 were in the maxilla. There were more unilocular than multilocular occurrences for the primary tumours, with a ratio of 2.2:1. These KCOTs were initially treated by simple enucleation (12 cases) or enucleation with Carnoy's solution (7 cases). After the initial surgery, 15 out of 19 (78.9%) recurred within 6 years, while 4 (21.1%) recurred after 6 years. Evidently, the recurrent lesion was involved with the roots of the teeth in three out of six cases whose teeth were preserved. In addition, the recurrent KCOTs had a tendency to be more multilocular or multifocal than the primary cases, with a unilocular-to-multilocular ratio of 1.1:1.
Conclusions
7.4% of primary KCOTs recurred within 6 years after initial treatment with either enucleation or a combination of enucleation and Carnoy's solution. The recurrent KCOTs were more likely to be multilocular or multifocal than the primary cases and often involved the teeth. The method of operation for these recurrent lesions would be considered as a more aggressive approach.
doi:10.1259/dmfr/22891281
PMCID: PMC3520371  PMID: 22301637
keratocystic odontogenic tumour; odontogenic keratocyst; enucleation; recurrence
13.  Dual functions of TAF7L in adipocyte differentiation 
eLife  2013;2:e00170.
The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.
DOI: http://dx.doi.org/10.7554/eLife.00170.001
eLife digest
The development of a single fertilized egg into a highly complex animal is determined by its genome, with a process called differential gene regulation exerting exquisite control over gene expression to ensure that various specialized cells are generated and that many types of tissue are produced. However, the mechanisms responsible for controlling gene expression and, therefore mammalian development, are poorly understood.
Researchers have developed a number of in vitro cell culture models to elucidate the details of differential gene regulation, and this approach has been used to characterize adipocytes—cells that store energy in the form of fat—for close to two decades. The formation of adipocytes, a process known as adipogenesis, has been extensively studied, but there remain major gaps in our knowledge: for example, the identities of many of the transcriptional regulators that are responsible for the differentiation of mesenchymal stem cells into adipocytes remain a mystery. This task is complicated by the fact that some of these regulators are involved in the differentiation of multiple cell lines, and that some of them also have multiple roles in the generation of a single cell type. In addition to being of fundamental interest, improving our knowledge of the properties and behavior of adipocytes is essential for tackling the increasing prevalence of obesity in the developed world.
Zhou et al. now report that TAF7L—a gene that was previously thought to be involved only in the production of sperm cells—has two roles in the differentiation of stem cells to form adipocytes. Using a combination of cellular, biochemical, genetic and genomic techniques, they show that TAF7L interacts with PPARγ, an important adipocyte transcriptional regulator at enhancer sites on the genome to increase the transcription of genes that are involved in adipogenesis. They also show that TAF7L interacts with a general transcription factor called TBP (short for TATA-binding protein) at promoter sequences, again to increase the expression of genes involved in adipogenesis. Moreover, they show that the expression of TAF7L in myoblasts—precursor cells that usually become muscle cells—can induce the formation of fat cells rather than muscle cells. Furthermore, mice lacking TAF7L are lean compared to their normal littermates. A clearer understanding of the underlying causes of fat cell formation could lead to the development of new approaches for the treatment of obesity and associated diseases.
DOI: http://dx.doi.org/10.7554/eLife.00170.002
doi:10.7554/eLife.00170
PMCID: PMC3539393  PMID: 23326641
ChIP-seq; RNA-seq; adipogenesis; C3H10T½; TAF7L; differentiation; Mouse
14.  Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity 
PLoS Genetics  2012;8(11):e1003038.
Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ∼3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA–deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity.
Author Summary
Small non-coding RNAs play critical roles during development and in disease. The integrity of the germline genome is of paramount importance to the wellbeing of offspring and the survival of species. Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs abundantly expressed in the gonad. Compared to microRNAs and small-interfering RNAs (siRNAs), the biogenesis and function of piRNAs remain poorly understood. Here we have identified MOV10L1, a putative RNA helicase, as a master regulator of piRNA biogenesis in mouse. We find that production of pachytene piRNAs requires MOV10L1. Blockade of pachytene piRNAs disrupts germ cell development and results in defects in post-meiotic genome integrity. Therefore, mutations in MOV10L1 and other piRNA pathway components may contribute to male infertility in humans.
doi:10.1371/journal.pgen.1003038
PMCID: PMC3499362  PMID: 23166510
15.  Disruption of Chtf18 Causes Defective Meiotic Recombination in Male Mice 
PLoS Genetics  2012;8(11):e1002996.
CHTF18 (chromosome transmission fidelity factor 18) is an evolutionarily conserved subunit of the Replication Factor C-like complex, CTF18-RLC. CHTF18 is necessary for the faithful passage of chromosomes from one daughter cell to the next during mitosis in yeast, and it is crucial for germline development in the fruitfly. Previously, we showed that mouse Chtf18 is expressed throughout the germline, suggesting a role for CHTF18 in mammalian gametogenesis. To determine the role of CHTF18 in mammalian germ cell development, we derived mice carrying null and conditional mutations in the Chtf18 gene. Chtf18-null males exhibit 5-fold decreased sperm concentrations compared to wild-type controls, resulting in subfertility. Loss of Chtf18 results in impaired spermatogenesis; spermatogenic cells display abnormal morphology, and the stereotypical arrangement of cells within seminiferous tubules is perturbed. Meiotic recombination is defective and homologous chromosomes separate prematurely during prophase I. Repair of DNA double-strand breaks is delayed and incomplete; both RAD51 and γH2AX persist in prophase I. In addition, MLH1 foci are decreased in pachynema. These findings demonstrate essential roles for CHTF18 in mammalian spermatogenesis and meiosis, and suggest that CHTF18 may function during the double-strand break repair pathway to promote the formation of crossovers.
Author Summary
Meiosis is the specialized process of cell division during germ cell development that results in formation of eggs and sperm. Genetic exchange between maternal and paternal chromosomes occurs during meiosis in a process called homologous recombination, in which DNA double- strand breaks are made and then repaired to allow DNA crossovers to form. These are essential processes that keep homologous chromosomes joined until anaphase I and ensure proper chromosome segregation. Errors in meiotic recombination lead to chromosome mis-segregation and ultimately aneuploidy, an abnormal chromosome number. Although it is well known that defects in these processes contribute greatly to infertility, birth defects, and pregnancy loss in humans, their molecular basis is not well understood. We demonstrate here a Chtf18 mutant mouse that exhibits subfertility and defects in meiotic recombination. Specifically, DNA double-strand breaks are incompletely repaired, DNA crossovers are significantly decreased, and homologous chromosomes separate during prophase I in Chtf18-null males. Our findings suggest roles for CHTF18 in DNA double-strand break repair and crossover formation, functions in mammals not previously known.
doi:10.1371/journal.pgen.1002996
PMCID: PMC3486840  PMID: 23133398
16.  Relating MEG Measured Motor Cortical Oscillations to resting γ-Aminobutyric acid (GABA) Concentration 
NeuroImage  2011;55(2):616-621.
The human motor cortex exhibits characteristic beta (15-30 Hz) and gamma oscillations (60-90 Hz), typically observed in the context of transient finger movement tasks. The functional significance of these oscillations, such as post-movement beta rebound (PMBR) and movement-related gamma synchrony (MRGS) remain unclear. Considerable animal and human non-invasive studies, however, suggest that the networks supporting these motor cortex oscillations depend critically on the inhibitory neurotransmitter γ-Aminobutyric acid (GABA). Despite such speculation, a direct relation between MEG measured motor cortex oscillatory power and frequency with resting GABA concentrations has not been demonstrated.
In the present study, motor cortical responses were measured from 9 healthy adults while they performed a cued button-press task using their right index finger. In each participant, PMBR and MRGS measures were obtained from time-frequency plots obtained from primary motor (MI) sources, localized using beamformer differential source localization. For each participant, complimentary magnetic resonance spectroscopy (MRS) GABA measures aligned to the motor hand knob of the left central sulcus were also obtained. GABA concentration was estimated as the ratio of the motor cortex GABA integral to a cortical reference NAA resonance at 2 ppm.
A significant linear relation was observed between MI GABA concentration and MRGS frequency (R2 = 0.46, p<0.05), with no association observed between GABA concentration and MRGS power. Conversely, a significant linear relation was observed between MI GABA concentration and PMBR power (R2 = 0.34, p<0.05), with no relation observed for GABA concentration and PMBR frequency. Finally, a significant negative linear relation between the participant’s age and MI gamma frequency was observed, such that older participants had a lower gamma frequency (R2 = 0.40, p < 0.05).
Present findings support a role for GABA in the generation and modulation of endogenous motor cortex rhythmic beta and gamma activity.
doi:10.1016/j.neuroimage.2010.12.077
PMCID: PMC3411117  PMID: 21215806
Magnetic Resonance Spectroscopy (MRS); γ-Aminobutyric acid (GABA); Event-related desynchrony / synchrony (ERD/ERS)
17.  Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma 
Blood Cancer Journal  2012;2(8):e82-.
Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK+ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK+ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK+ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2active cells, but not Sox2inactive cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK+ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.
doi:10.1038/bcj.2012.27
PMCID: PMC3432482  PMID: 22885405
Sox2; transcriptional activity; NPM-ALK; STAT3; tumorigenicity
18.  Ameloblastic fibro-odontosarcoma of the mandible: imaging findings 
Wang, S | Shi, H | Wang, P | Yu, Q
Dentomaxillofacial Radiology  2011;40(5):324-327.
We report a histologically verified case of ameloblastic fibro-odontosarcoma (AFOS) affecting the right mandible. CT scanning showed an expansile intraosseous mass with ill-defined borders and slightly dense opacities.
doi:10.1259/dmfr/80061108
PMCID: PMC3520256  PMID: 21697160
jaw; ameloblastic, fibro-odontosarcoma; panoramic radiograph; computed tomography imaging
19.  Detection of dental root fractures by using cone-beam computed tomography 
Dentomaxillofacial Radiology  2011;40(5):290-298.
Objectives
The purpose of this study was to compare the diagnostic accuracy of cone beam CT (CBCT) with that of conventional dental radiography in the detection of root fractures and to evaluate the influence of root canal fillings on root fracture detection.
Methods
We investigated 128 patients with clinically suspected root fractures in 135 teeth. These patients underwent conventional dental radiography, CBCT and eventually surgical exploration. Among the 135 teeth, 86 were non-endodontically treated teeth and 49 were endodontically treated teeth. Two oral radiologists independently analysed the dental radiographs and CBCT images of each patient and reached a consensus. The CBCT findings of root fractures were set as the detection of a separation of the adjacent root segments on at least two contiguous sections and on at least two of the three-dimensional (3D) planes.
Results
Root fracture was intraoperatively detected in 95 of the 135 teeth. The sensitivity and specificity of root fractures diagnosed on the basis of the consensus between the 2 evaluators were 26.3% and 100%, respectively, for dental radiography and 89.5% and 97.5%, respectively, for CBCT. CBCT was significantly more accurate than dental radiography in detecting root fractures (P < 0.001). The sensitivity of CBCT was reduced in the presence of root canal fillings but its specificity remained unaffected. Both the sensitivity and specificity of dental radiography were not influenced by the presence of root canal fillings.
Conclusions
CBCT appears to be more accurate than conventional dental radiography in the detection of root fractures.
doi:10.1259/dmfr/84907460
PMCID: PMC3520261  PMID: 21697154
cone-beam computed tomography; dental radiography; diagnosis; dental root fracture
20.  Secondary chondrosarcoma in the mandibular condyle 
Xu, B | Shi, H | Wang, S | Wang, P | Yu, Q
Dentomaxillofacial Radiology  2011;40(5):320-323.
We report the case of a woman who had secondary chondrosarcoma in an osteochondroma in the left mandibular condyle, presenting as a large lobulated bone density mass in the left condyle process, to illustrate the clinical characteristics and imaging findings.
doi:10.1259/dmfr/17575463
PMCID: PMC3520265  PMID: 21697159
secondary chondrosarcoma; mandibular condyle; imaging
21.  Evaluation of supporting role of a foldable capsular vitreous body with magnetic resonance imaging in the treatment of severe retinal detachment in human eyes 
Zhang, R | Wang, T | Xie, C | Lin, X | Jiang, Z | Wang, Z | Liu, Y | Luo, Y | Long, C | He, L | Wang, P | Gao, Q
Eye  2011;25(6):794-802.
Purpose
To determine the supporting role of a novel foldable capsular vitreous body (FCVB) with magnetic resonance imaging (MRI) in the treatment of severe retinal detachment in human eyes.
Methods
The study examined nine eyes of nine patients. Among the nine eyes, five had suffered penetrating injuries while four had suffered contusions of the eyeball involving large defects of the retina or choroids. A standard three-port pars plana vitrectomy was performed, FCVB was triple-folded and sent into the vitreous cavity; balanced salt solution (BSS) was injected into the capsule to support the retina. Three cardinal axes of nine eyes were examined using MRI at baseline and at the 3-month follow up.
Results
MRI revealed that the signal intensity of the FCVB was similar to the normal vitreous body, with low-signal intensity on T1-weighted image and high-signal intensity on T2-weighted image. In three pre-operative silicone oil- or heavy silicone oil-filled eyes, FCVBs were not fully inflated, and eyeball deformation was observed in one eye. Shifts of three cardinal axes of three eyes (horizontal, anteroposterior, and vertical) according to MRI, were −4.33, −4.67, and −2.67 mm. In the remaining six eyes, FCVBs were well distributed in the vitreous cavity and evenly supported the retina; the cardinal axes of the eyes were similar to pre-operation. Shifts of three cardinal axes of six eyes were −0.34, −0.34, and −0.34 mm. In a total of nine eyes, shifts of three cardinal axes were −1.67, −1.77, and −1.11 mm. Statistically significant difference showed only between the horizontal axis of nine eyes pre-operatively and post-operatively (P1=0.041, P2=0.058, P3=0.123).
Conclusion
This study demonstrated the effectiveness of MRI to monitor the supporting role of an FCVB in the treatment of severe retinal detachment in human eyes.
doi:10.1038/eye.2011.61
PMCID: PMC3178141  PMID: 21423138
FCVB; human eyes; magnetic resonance imaging
22.  Nxf3 is Expressed in Sertoli Cells, but is Dispensable for Spermatogenesis 
SUMMARY
In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase.
doi:10.1002/mrd.21291
PMCID: PMC3081378  PMID: 21308854
Sertoli cells; testis; spermatogenesis; Nxf3
23.  MNS1 Is Essential for Spermiogenesis and Motile Ciliary Functions in Mice 
PLoS Genetics  2012;8(3):e1002516.
During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1–deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1–deficient sperm flagella, the characteristic arrangement of “9+2” microtubules and outer dense fibers are completely disrupted. In addition, MNS1–deficient mice display situs inversus and hydrocephalus. MNS1–deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.
Author Summary
Cilia are microtubule-based structures present in virtually all cells in vertebrates. Cilia have diverse functions in development, growth, signaling, and fertilization. Primary ciliary dyskinesia (PCD) affects one in 16,000 individuals. PCD is characterized by bronchiectasis and chronic sinusitis, and is often associated with situs inversus and male infertility. The genetic cause of PCD is heterogeneous. Some cases of PCD in humans and animals are caused by single genic mutations such as mutations in genes encoding microtubule-based dynein arm components. We have characterized a protein called MNS1 and found that it plays an essential role in ciliary functions in mice. MNS1 is a novel and integral component of sperm flagella. Mice lacking MNS1 exhibit male sterility as evidenced by abnormal assembly of sperm flagella. MNS1–deficient mice also display defects in left–right asymmetry patterning of internal organs and hydrocephalus. Therefore, mutations in MNS1 may contribute to male infertility and PCD in humans.
doi:10.1371/journal.pgen.1002516
PMCID: PMC3291534  PMID: 22396656
24.  Muscarinic Acetylcholine Receptor Subtypes Mediating Urinary Bladder Contractility and Coupling to GTP Binding Proteins 
Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.
PMCID: PMC3277792  PMID: 7752101
25.  Lafora bodies in skeletal muscle are fiber type specific 
Neurology  2011;76(19):1674-1676.
doi:10.1212/WNL.0b013e318219faf6
PMCID: PMC3100090  PMID: 21451149

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