TAF5L and TAF6L Maintain Self-Renewal of Embryonic Stem Cells via the MYC Regulatory Network.

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.

Reduction in Offset Is Associated With Worse Functional Outcomes Following Total Hip Arthroplasty.

BACKGROUND: Conflicting reports exist about the effect of offset variation on functional outcomes following total hip arthroplasty. Reproducing native hip offset is thought to optimize function by restoring biomechanics and appropriately tensioning the hip abductor muscles. The aim of this study is to assess the effect of failing to restore global hip offset in comparison to the native contralateral hip. METHODS: A retrospective analysis of a prospective patient cohort was performed on patients undergoing an elective primary total hip arthroplasty. A total of 414 patients who had a minimum of 12 months of follow-up were included. Postoperative plain radiographs were analyzed for offset and compared to the contralateral native hip. Western Ontario and McMaster Universities Arthritis Index (WOMAC) and Veterans RAND 12 (VR-12) scores were assessed preoperatively and at 12 months postoperatively. RESULTS: Regression analyses indicated that a reduction in offset of >20 mm resulted in worse WOMAC pain (P = .005) and motion (P = .015) scores compared to those with maintained offset. WOMAC function (P = .063), global (P = .025), and VR-12 scores were not affected (physical P = .656; mental P = .815). Reduction in offset up to 20 mm and increased offset were not significantly associated with patient-reported outcome measures (P-values ranged from .102 to .995). CONCLUSION: This study demonstrated an association between reduction in offset by >20 mm and worse WOMAC pain and motion scores following total hip arthroplasty. Surgeons should avoid decreases in offset >20 mm in order to optimize functional outcomes.

A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in murine ESCs.

Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elements within the ∼12-kb cis-region (cis-REs; CREs) of the Oct4 gene locus, as well as genome-wide 2,613 high-confidence trans-REs (TREs), in mESCs. ctSCAN-SMS identified 10 CREs and 12 TREs as novel candidate REs of the Oct4 gene in mESCs. Furthermore, deletions of these candidate REs confirmed that the majority of the REs are functionally active, and CREs are more active than TREs in controlling Oct4 gene expression. A subset of active CREs and TREs physically interact with the Oct4 promoter to varying degrees; specifically, a greater number of active CREs, compared with active TREs, physically interact with the Oct4 promoter. Moreover, comparative genomics analysis reveals that a greater number of active CREs than active TREs are evolutionarily conserved between mice and primates, including humans. Taken together, our study demonstrates the reliability and robustness of ctSCAN-SMS screening to identify critical REs and investigate their roles in the regulation of transcriptional output of a target gene (in this case Oct4) in their native context.

Effect of Polyester and Plaster of Paris Casts on Determination of Volumetric Bone Mineral Density Assessed by Peripheral Quantitative Computed Tomography (pQCT).

Peripheral quantitative computed tomography (pQCT) is a non-invasive, low-radiation tool for measuring volumetric bone mineral density. It has potential for use in fracture healing applications; however, the unknown attenuation effects of cast material on peripheral quantitative computed tomography have contributed to its limited use in this area. The effect of two common cast materials, polyester and Plaster of Paris was investigated by performing both in vitro and in vivo studies. The in vitro study tested the effect of increasing layers of cast material on bone density measurements performed on a hydroxyapatite phantom. Cast thickness was directly associated with a reduction in bone mineral density, with twelve layers of polyester and Plaster of Paris resulting in a 0.55 and 2.21 % decrease in bone density measurements. Precision error in situ with polyester cast material was 0.71 %, and 2.31 % with Plaster of Paris cast material. The in vivo study comprised a prospective trial with 28 healthy adult participants to evaluate the effect of the two cast materials. Trabecular bone mineral density was increased by 0.5 % in the presence of a polyester cast and decreased by 4.22 % in the presence of a Plaster of Paris cast. Cortical bone mineral density was decreased by 3.46 and 5.54 % for polyester and Plaster of Paris, respectively. This study quantified the effects of orthopaedic casts on pQCT-derived bone parameters. The results suggest applicability of commonly utilised cast materials in combination with pQCT to assess fracture healing.

Incidence of Complex Regional Pain Syndrome I Following Foot and Ankle Fractures Using the Budapest Criteria.

OBJECTIVE : Fractures are a well-recognized inciting event in the development of complex regional pain syndrome. This study aimed to prospectively determine the incidence of complex regional pain syndrome following foot and ankle fractures. METHODS : A prospective study was conducted of patients presenting to two metropolitan hospitals with plain radiograph diagnosis of fractures to the foot or ankle. Patients were initially screened by phone 3 months after injury using the validated International Association for the Study of Pain Budapest criteria. Patients who fulfilled the screening criteria were then physically examined by a pain specialist to assess clinical signs as part of the Budapest criteria. RESULTS : A total of 306 consecutive eligible patients were included. One hundred and ten patients reported at least one symptom of complex regional pain syndrome; however, only three fulfilled the minimum requirements to necessitate clinical review. Of these three, only one patient fulfilled the combination of symptom and sign criteria for a positive diagnosis according to the validated Budapest criteria. The incidence of complex regional pain syndrome following foot and ankle fracture in this study was 0.3%. CONCLUSION : Although many patients may experience vasomotor, sensory, and sudomotor disturbance following a fracture to the foot and ankle, the observed incidence of complex regional pain syndrome using a prospectively collected validated criteria is significantly lower than previously published.

Vitamin E supplementation and mortality in healthy people: a meta-analysis of randomised controlled trials.

PURPOSE: To evaluate the effect of oral vitamin E supplementation on all-cause mortality in apparently healthy people. METHODS: A systematic review and meta-analysis was conducted on randomised controlled trials (RCTs) with ≥ 6 months of follow up investigating the effect of vitamin E supplementation on healthy adults in developed countries. Electronic databases (MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials) and reference lists of trial reports were searched for RCTs published between 1966 and June 2012. Three investigators assessed eligibility of identified trials. Disagreements were resolved by consensus. Two investigators independently extracted data according to the criteria. RESULTS: There were 18 RCTs identified with 142,219 apparently healthy participants (71,116 in vitamin E intervention groups and 71,103 in control groups) that were included in the final analysis. Fixed effect and random effects analysis of the 18 trials revealed that supplementation with vitamin E was not associated with all-cause mortality (relative risk 1.01, 95% confidence interval 0.97 - 1.05, p = 0.65). Subgroup analyses by type of vitamin E (natural or synthetic), dose or duration of exposure, study design or quality, and pre-specified mortality outcome showed no association with all-cause mortality. CONCLUSIONS: The evidence from pooled analysis of 18 randomised controlled trials undertaken in apparently healthy people shows no effect of vitamin E supplementation at a dose of 23-800 IU/day on all-cause mortality.

The apparent loss of PRC2 chromatin occupancy as an artifact of RNA depletion.

RNA has been implicated in the recruitment of chromatin modifiers, and previous studies have provided evidence in favor and against this idea. RNase treatment of chromatin is commonly used to study RNA-mediated regulation of chromatin modifiers, but the limitations of this approach remain unclear. RNase A treatment during chromatin immunoprecipitation (ChIP) reduces chromatin occupancy of the H3K27me3 methyltransferase Polycomb repressive complex 2 (PRC2). This led to suggestions of an "RNA bridge" between PRC2 and chromatin. Here, we show that RNase A treatment during ChIP causes the apparent loss of all facultative heterochromatin, including both PRC2 and H3K27me3 genome-wide. We track this observation to a gain of DNA from non-targeted chromatin, sequenced at the expense of DNA from facultative heterochromatin, which reduces ChIP signals. Our results emphasize substantial limitations in using RNase A treatment for mapping RNA-dependent chromatin occupancy and invalidate conclusions that were previously established for PRC2 based on this assay.

Comparison of the moulding ability of Plaster of Paris and polyester cast material in the healthy adult forearm.

OBJECTIVES: To quantify the moulding ability of Plaster of Paris and polyester cast materials as assessed by the novel use of peripheral quantitative computed tomography. METHODS: A prospective crossover study was performed in 25 healthy volunteers aged 18-65 years. Participants' non-dominant wrist was immobilized using a synthetic polyester cast followed by a Plaster of Paris cast with three point moulding to simulate reduction of a dorsally angulated distal radius fracture. The novel use of peripheral quantitative computed tomography was used to measure the closeness of fit of each cast on an axial tomographic slice. RESULTS AND CONCLUSIONS: Plaster of Paris casts were able to achieve a closer mould than polyester when measured between the bone and the cast (p=0.002), as well as between the skin and the cast (p=0.001). There was no difference when stratified on BMI. Using pQCT assessment, a closely moulded fit was able to be more consistently achieved when using Plaster of Paris when compared to polyester casts of the distal radius. LEVEL OF EVIDENCE: III.

Effects of small pulsed nanocurrents on cell viability in vitro and in vivo: implications for biomedical electrodes.

Using a custom-built, implantable pulse generator, we studied the effects of small pulsed currents on the viability on rat aortic-derived cells (RAOC) in vitro. The pulsed currents (0.37A/m(2)) underwent apoptosis within 24h as shown by the positive staining for cleaved caspase-3 and classically apoptotic morphology. Based on these findings, we examined the effects of nanocurrents in vivo. The pulse generator was implanted subcutaneously in the rat model. The electrode|tissue interface histology revealed no difference between the active platinum surface and the neighboring control surface, however we found a large difference between electrodes that were functional during the entire experiment and non-active electrodes. These non-active electrodes showed an increase in impedance at higher frequencies 21 days post-implantation, whereas working electrodes retained their impedance value for the entire experiment. These results indicate that applied currents can reduce the impedance of implanted electrodes.

Increased prevalence of interstitial cystitis: previously unrecognized urologic and gynecologic cases identified using a new symptom questionnaire and intravesical potassium sensitivity.

OBJECTIVES: Most individuals with interstitial cystitis (IC) have both pelvic pain and urinary urgency/frequency, and many have dyspareunia. Existing questionnaires designed to assess bladder-origin pelvic pain (IC) give little attention to pelvic pain or dyspareunia, however. On the basis of our clinical experience with more than 5000 patients with IC, we have designed a pelvic pain and urgency/frequency (PUF) symptom scale that gives balanced attention to urinary urgency/frequency, pelvic pain, and symptoms associated with sexual intercourse. METHODS: We used the intravesical potassium sensitivity test (PST) to validate the PUF scale in urologic patients suspected of having IC, gynecologic patients with pelvic pain, controls, and women attending lectures given by one of us (C.L.P.). Positive potassium sensitivity is known to be associated with a bladder epithelial dysfunction present in most individuals with IC. RESULTS: The PST was positive in 74% of patients with a PUF score of 10 to 14, 76% of those scoring 15 to 19, and 91% of those scoring 20 or higher. All controls' PUF scores were less than 3, and the rate of positive PST in controls was 0%. The PUF scores in women screened at lectures suggested that 1 in 4.5 women have IC. CONCLUSIONS: High PUF scores appear to correlate directly with a higher likelihood of positive PST in both urologic patients suspected of having IC and gynecologic patients with pelvic pain. The PUF appears to be a valid tool for detecting IC in these two populations, as well as in the general population. Use of the PUF alone may prove to be an accurate method for detecting IC. The IC prevalence may be as high as 1 in 4.5 women.

The prevalence of interstitial cystitis in gynecologic patients with pelvic pain, as detected by intravesical potassium sensitivity.

OBJECTIVE: The purpose of this study was to determine the prevalence of interstitial cystitis in a large number of gynecologic patients with pelvic pain versus control subjects, as indicated by a positive result on a potassium sensitivity test. STUDY DESIGN: Gynecologists at four US medical centers administered the potassium sensitivity test to consecutive unselected patients with pelvic pain and control subjects. Before testing, each patient with pelvic pain was given an initial clinical diagnosis on the basis of the chief symptomatic complaint(s) and was surveyed for urologic symptoms. RESULTS: Of 244 patients with pelvic pain, 197 patients (81 %) had a positive result from a potassium sensitivity test. Positive potassium sensitivity test rates were comparable across all four sites and all clinical diagnoses that included endometriosis, vulvodynia (vulvar vestibulitis), and pelvic pain. Urologic symptoms were reported by 84% of patients, but only 1.6% of the patients had received an initial diagnosis of interstitial cystitis. None of the 47 control subjects were tested positive with the potassium sensitivity test. CONCLUSION: Interstitial cystitis may be a common unrecognized cause of pelvic pain in gynecologic patients and deserves greater, if not primary, consideration in the differential diagnosis of pelvic pain.