Computer-aided engineering of stabilized fibroblast growth factor 21.

FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

EURO-MUSCULUS/USPRM Dynamic Ultrasound Protocols for Ankle/Foot.

ABSTRACT: In this dynamic scanning protocol, ultrasound examination of the ankle is described using various maneuvers to assess different conditions. Real-time patient examination and scanning videos are used for better simulation of daily clinical practice. The protocol is prepared by several/international experts in the field of musculoskeletal ultrasound and within the umbrella of European Musculoskeletal Ultrasound Study Group in Physical and Rehabilitation Medicine/Ultrasound Study Group of the International Society of Physical and Rehabilitation Medicine.

Injury to the posterior malleolus in Maisonneuve fractures.

PURPOSE: The aim of this study was to describe the incidence and a complex pathoanatomy of posterior malleolus fractures in a Maisonneuve fracture. METHODS: The study included 100 prospectively collected patients with a complete clinical and radiological documentation of an ankle fracture or fracture-dislocation including a fracture of the proximal quarter of the fibula. RESULTS: A posterior malleolus fracture was identified in 74 patients, and in 27% of these cases it carried more than one quarter of the fibular notch. Displacement of the posterior fragment by more than 2 mm was shown by scans in 72% of cases. Small intercalary fragments were identified in 43% of cases. Fractures of the Tillaux-Chaput tubercle were identified in 20 patients. CONCLUSION: Our study has proved a high rate of posterior malleolus fractures associated with a Maisonneuve fracture, and documented their considerable variability in terms of involvement of the fibular notch, tibiotalar contact area, direction of displacement and frequency of intercalary fragments. Of no less importance is a combination of Tillaux-Chaput fractures with a Maisonneuve fracture.

Cerebral organoids derived from patients with Alzheimer's disease with PSEN1/2 mutations have defective tissue patterning and altered development.

During the past two decades, induced pluripotent stem cells (iPSCs) have been widely used to study human neural development and disease. Especially in the field of Alzheimer's disease (AD), remarkable effort has been put into investigating molecular mechanisms behind this disease. Then, with the advent of 3D neuronal cultures and cerebral organoids (COs), several studies have demonstrated that this model can adequately mimic familial and sporadic AD. Therefore, we created an AD-CO model using iPSCs derived from patients with familial AD forms and explored early events and the progression of AD pathogenesis. Our study demonstrated that COs derived from three AD-iPSC lines with PSEN1(A246E) or PSEN2(N141I) mutations developed the AD-specific markers in vitro, yet they also uncover tissue patterning defects and altered development. These findings are complemented by single-cell sequencing data confirming this observation and uncovering that neurons in AD-COs likely differentiate prematurely.

Administration of maximum dose of mucolytic solution before upper endoscopy - a double-blind, randomized trial.

OBJECTIVE: The aim of the study was to demonstrate that an administration of mucolytic solution with a maximum dose of simethicone and n -acetylcysteine before upper endoscopy improves mucosal visibility compared to a group without administration of mucolytic solution or water. METHODS: This study was a double-blind, randomized controlled trial. Patients were randomized into four groups, with the administration of 100 ml of water + 600 mg n -acetylcysteine + 400 mg simethicone, 100 ml of water + 400 mg n -acetylcysteine + 20 mg simethicone, 100 ml of water, and without any water or mucolytic solution. During the examination, a total of 10 images were taken in the defined areas. The overall visibility score was given by the sum of the 0-5 scores of the five areas and was assessed by the endoscopist performing the procedure and the blinded endoscopists using static images. RESULTS: A total of 129 patients were randomized. The group of patients did not differ in age, sex distribution, and indications significantly. The overall visibility score as assessed by the endoscopist performing the procedure was significantly higher in the group with the maximum dose of mucolytic solution compared to the group without solution or water (18.9 ±â€…2.9 vs. 16.6 ±â€…3.3, P  = 0.023). This difference was not evident by the blinded evaluation of static photographs. CONCLUSION: Administration of mucolytic solution with a maximum dose of n -acetylcysteine and simethicone before upper endoscopy improved mucosal visibility in the upper gastrointestinal tract compared with the group without any preparation while evaluated by performing endoscopist.

Lateral malleolar crest and its clinical importance.

PURPOSE: During study of anatomy of a fractured posterior malleolus of the ankle on CT scans, the authors noticed a prominent crest on the lateral malleolus, which they termed the lateral malleolar crest (LMC). As, in their view, LMC is a clinically important structure which was only briefly mentioned by a few authors without an official term, they focused on the anatomy of this structure. MATERIALS AND METHODS: A total of 352 dry fibulae were analyzed and the following parameters recorded: (F) length of the fibula, (LMC) total length of LMC, (A) length of the part of the examined crest from the superior border of the articular facet of the lateral malleolus (AFLM) to its most proximal intersection with the midline of the fibula, (B) height of the medial triangular rough surface, and (A/F) A/F ratio. RESULTS: The crest was observed in all specimens. (F) was 346.5 ± 26 mm (95% confidence interval [CI] 344-349), (LMC) was 85.4 ± 11.6 mm (95% CI 84.2-86.6), (A/F) was 25% ± 3% (95% CI 24.7-25.3) in the whole group. (A) was 25.9 ± 6.5 mm (95% CI 24.8-26.8) in the whole group, (B) was 34.9 ± 4.7 mm (95% CI 34.3-35.5) in the whole group, 36 ± 6.1 mm (95% CI 35.1-36.9). CONCLUSION: LMC is an important structure on the lateral malleolus. The knowledge of its anatomy is essential for placement of syndesmotic screws or/and the fibular plate.

Administration of mucolytic solution before upper endoscopy - double-blind, monocentric, randomized study.

AIMS: Sufficient visibility of the mucosa during upper endoscopy is crucial for successful diagnosis, especially for early neoplastic lesions. Data documenting the effect of administration of mucolytic solution prior to gastroscopy in order to improve mucosal visibility are limited in Europe. The aim of the study was to assess the score of mucosal visibility in the upper gastrointestinal tract after administration of the mucolytic solution defined by us. PATIENTS AND METHODS: This is a monocentric, double-blind, randomized study involving 134 patients indicated for diagnostic upper endoscopy. Patients were randomly assigned to one of three arms, with mucolytic solution (100 mL water + 400 mg N-acetylcysteine + 20 mg simethicone), without the solution , and with 100 mL pure water. During the examination, 11 photographs were taken in defined areas. The visibility score was given by the sum of the score 0-5 from 5 defined localities evaluated by a blinded endoscopist and subsequently by two blinded endoscopists. Other parameters monitored were examination time and a semiquantitative evaluation of residual gastric fluid. RESULTS: The basic characteristics of the group (sex, age, indications for examination) were comparable between arms. The visibility score was similar in all arms - 17.4 ± 1.9 vs. 17.0 ± 2.0 vs. 17.6 ± 1.8 (P=0.32). The examination time and the amount of residual fluid in the stomach were comparable in all arms. CONCLUSIONS: Administration of the mucolytic solution in our study did not increase the mucosal visibility score in the esophagus, stomach and duodenum. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02967094.

Faecal microbiota transplantation in the treatment of recurrent intestinal Clostridioides difficile infection - a ten-year single-center experience.

Clostridioides difficile (Clostridium difficile in older taxonomy) is a gram-positive anaerobic and bacteria enabled by endospores. Clostridioides difficile is currently the main cause of nosocomial infections in developed countries. Due to the high probability of developing bacterial resistance to treatment and the numerous recurrences in multiple chronic conditions in older adults of our society it causes a widespread medical problem. Faecal microbiota transplantation (FMT) is a highly effective method for treating recurrent intestinal Clostridioides difficile infections (CDI). With this method the potential mechanism of effect is the transmission of a complex intestinal ecosystem, including vital microorganisms, from the donor to the recipient. Presenting the results of monocentric prospective monitoring: Primary aim of the study was to evaluate long-term remission (the continued absence of clinical manifestations of CDI 3 months after FMT administration). The secondary aim of the study was to monitor the short-term remission in the 7 days after FMT administration. Demographic data, information about CDI and the details of therapy were obtained and completed by the treating physician of each patient or by targeted questioning of the patient or their family. We used clinical monitoring to determine the effect of the treatment. The examinations of stool donors and the preparation for a faecal microbiota transplantation were performed according to the currently valid guidelines of the Czech Society of Infectious Diseases for the treatment of the recurrent bacterial infection Clostridioides difficile with faecal microbiota transplantation. The follow-ups took place from February 2011 to July 2021 in the gastroenterology department at the AGEL Ostrava-Vítkovice Hospital and included 116 patients with their first and subsequent recurrence of CDI that were treated with faecal bacteriotherapy. The median age of our patients was 71 years old (the youngest was 19 years old, the oldest 103 years old). 69 women and 47 men took part in the study. 56 patients had their first recurrence of CDI, 41 had a second attack, and 20 patients had a third and subsequent recurrences. In 62 patients (53.4 %), the route of FMT administration was a local enema into the left colon. With 37 patients (31.9 %) we used a colonoscopy after standard anterograde bowel preparation. With 12 patients (10.3 %) gastroscopy administration (deep into the duodenum) was used. 4 patients (3.5 %) were given a nasoenteral tube and one patient (0.9 %) was administered FMT per percutaneous endoscopic gastrostomy (PEG). We applied a frozen universal donor FMT in 81 patients (69.8 %), and a freshly prepared FMT from a person living in the same household was used in 35 patients (30.1 %). The secondary endpoint (the absence of clinical manifestations of CDI within 7 days of FMT administration) was achieved with 102 patients (87.9 %) in our study. The fulfilment of the primary endpoint (the development of long-term remission) was observed with 93 patients (80.2 %). An early administration of FMT appears to be a significant predictor of treatment effect (p = 0.05; OR 5.11; 95% CI 1.65-15.8). Faecal microbiota transplantation is an effective and safe therapy for recurrent intestinal Clostridioides difficile infection, and it respects the up-to-date guidelines for treatment. Of the 116 patients included in our study with first and subsequent CDI, we achieved long-term remission in 80.2 % of them. An early administration of FMT appears to be a significant predictor of treatment effect.

Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations.

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. Although uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques that involve averaging of cell populations or analysis of clusters of tandem repeats in a single-cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context, and thus constitutes an important tool for gaining a complete understanding of the epigenetic organization of genomes. We report that using an optimized fiber extension protocol is essential in order to obtain more reproducible data and to minimize the clustering of fibers. We also demonstrate that the use of super-resolution microscopy is important for reliable evaluation of the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script for the analysis of methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart.

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.

Pediatric dens anatomy and its implications for fracture treatment: an anatomical and radiological study.

PURPOSE: Separation of C2 growth plates and dens fractures are the most common types of injuries to the axis (C2) in children. Operative treatment of these injuries with the use of direct osteosynthesis requires a profound knowledge of detailed anatomy and dimensions of the axis. The main issue addressed by the study was the age at which the size of the dens is adequate at all levels to accommodate two screws, and the size of the posterior dens angulation angle (PDAA) in a healthy child in individual age periods. METHODS: Dimensions and angles of the dens and C2 in individual age categories in both boys and girls were measured in a series of 203 CT scans of individuals 0-18 years old and on anatomical specimens (42 samples). In addition, 5 histological series of this region from the fetal period were reviewed. RESULTS: Dimensions of the dens gradually increase with age, with a considerable acceleration during growth spurt periods that are different in boys and girls. PDAA is markedly changing with age; in the fetal period, the dens shows a slight anterior angulation which gradually transforms into posterior angulation, as early as between 4 and 6 years of age. The screw insertion angle changes accordingly. CONCLUSION: During growth, there occur changes in PDAA that should be respected in evaluation of transformation of anterior into posterior angulation, as shown by imaging methods. Dens dimensions theoretically allow insertion of two 3.5 mm screws as early as from the age of 1 year.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells.

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

The fibular notch: an anatomical study.

INTRODUCTION: Reduction of the distal fibula into the fibular notch (FN) poses a problem that has not been fully resolved, yet. A number of methods have been developed for the assessment of the position of the fibula in the FN, but none of them is ideal. A majority of authors assess the FN 1 cm above the tibiotalar joint space, without specifying the reason for the choice of this distance. None of the previous studies has addressed at what level the FN is the deepest. Our findings show that it is 4-5 mm above the ankle joint space and verification of this hypothesis has been the aim of this study. MATERIALS AND METHODS: Dry adult tibial bone specimens from the Pachner's collection of the Institute of Anatomy of 1st Faculty of Medicine, Charles University, Prague were used in the study. Height of the FN at its widest point, 3 mm and 10 mm above the articular surface of the distal tibia were measured in each specimen, as well as the depth of the FN at the deepest point, 3 mm and 10 mm above the articular surface of the distal tibia and the distance between the highest point of this surface and the deepest point of the notch. RESULTS: The mean length of the tibia was 350 mm; the mean height of the FN was 42.5 mm; the mean width of the FN at its widest point was 23.6 mm, at 3 mm above the tibiotalar joint space 22 mm, 10 mm above this articular surface of distal tibia (tibial plafond) 18.9 mm. The mean depth of the notch at 3 mm above the tibial plafond was 3.8 mm, at 10 mm above this surface 4.1 mm. The maximum mean depth of the notch was 4.5 mm, the distance from this point to the highest point of the tibial plafond was 5.3 mm. CONCLUSION: The deepest point of the FN lies 5 mm above the articular surface of the tibial plafond, with the mean value of the depth being 4.5 mm. This region is, therefore, ideal for assessment of the position of the distal fibula in the FN.

Both Hypoxia-Inducible Factor 1 and MAPK Signaling Pathway Attenuate PI3K/AKT via Suppression of Reactive Oxygen Species in Human Pluripotent Stem Cells.

Mild hypoxia (5% O2) as well as FGFR1-induced activation of phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) and MAPK signaling pathways markedly support pluripotency in human pluripotent stem cells (hPSCs). This study demonstrates that the pluripotency-promoting PI3K/AKT signaling pathway is surprisingly attenuated in mild hypoxia compared to the 21% O2 environment. Hypoxia is known to be associated with lower levels of reactive oxygen species (ROS), which are recognized as intracellular second messengers capable of upregulating the PI3K/AKT signaling pathway. Our data denote that ROS downregulation results in pluripotency upregulation and PI3K/AKT attenuation in a hypoxia-inducible factor 1 (HIF-1)-dependent manner in hPSCs. Using specific MAPK inhibitors, we show that the MAPK pathway also downregulates ROS and therefore attenuates the PI3K/AKT signaling-this represents a novel interaction between these signaling pathways. This inhibition of ROS initiated by MEK1/2-ERK1/2 may serve as a negative feedback loop from the MAPK pathway toward FGFR1 and PI3K/AKT activation. We further describe the molecular mechanism resulting in PI3K/AKT upregulation in hPSCs-ROS inhibit the PI3K's primary antagonist PTEN and upregulate FGFR1 phosphorylation. These novel regulatory circuits utilizing ROS as second messengers may contribute to the development of enhanced cultivation and differentiation protocols for hPSCs. Since the PI3K/AKT pathway often undergoes an oncogenic transformation, our data could also provide new insights into the regulation of cancer stem cell signaling.

Endosonography-Guided Fine-Needle Aspiration versus "Key-Hole Biopsy" in the diagnostics of upper gastrointestinal subepithelial tumors. A prospective randomized interventional study.

BACKGROUND: The management and prognosis of subepithelial tumors (SETs) of the upper gastrointestinal tract depend on the correct preoperative evaluation, including tissue diagnosis in selected cases. Several methods providing deep tissue sampling for cytological and/or histological examinations have been described but their diagnostic yield and precise position in the diagnostic algorithm remain to be established. This prospective randomized study aims to compare the Endosonography-Guided Fine-Needle Aspiration (EUS-FNA) to Key-Hole Biopsy (KHB) in cytological or histological diagnostics of upper gastrointestinal SETs. PATIENTS AND METHODS: This study was conducted in a single tertiary endoscopy center in Ostrava, Czech Republic between November 2010 and October 2015. Patients with endoscopically detected SETs of the upper gastrointestinal tract with a diameter ≥ 2 cm, were randomized to either the EUS-FNA with 22G needle, or to the Key Hole biopsy (forceps biopsy through mucosal incision) groups. The main study outcomes were success rate of tissue diagnostics and, in the cases of Gastrointestinal Stromal Tumours (GIST), possibility of determining mitotic activity. A cross-over examination was performed in situations where the first method had failed. RESULTS: A total of 46 consecutive patients were randomized. Of these, 24 (52%) and 22 (48%) were randomized to EUS-FNA group and KHB arm, respectively. 5 SETs (11%) were detected in the esophagus, 40 (87%) in the stomach and 1 (2%) in the duodenum. The definitive diagnosis was established by the first sampling method in 42 (91%) patients, including 22 (92%) in the EUS-FNA group and 20 (91%) in the KHB group (P=0.999), and after a cross-over in another 3 (7%) patients. The most prevalent SET was GIST (70%). Although some mitotic activity could be observed in 11 patients, the mitotic index could be diagnosed in none of them. Of a total of 20 surgically treated patients, preoperative and postoperative tissue diagnosis corresponded in 19/20 (95%) cases, including 100% in FNA group and 91% in KHB group (P=0.999). No adverse events of tissue sampling occurred in the study. CONCLUSIONS: Deep tissue sampling by EUS-FNA and KHB are equally effective in the diagnostics of SETs of the upper gastrointestinal tract ≥ 2 cm. However, neither EUS-FNA nor KHB provided adequate tissue sample to determine mitotic index. TRIAL REGISTRATION: Clinicaltrials.gov (NCT02025244).

Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raska, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

Dystrophin Deficiency Leads to Genomic Instability in Human Pluripotent Stem Cells via NO Synthase-Induced Oxidative Stress.

Recent data on Duchenne muscular dystrophy (DMD) show myocyte progenitor's involvement in the disease pathology often leading to the DMD patient's death. The molecular mechanism underlying stem cell impairment in DMD has not been described. We created dystrophin-deficient human pluripotent stem cell (hPSC) lines by reprogramming cells from two DMD patients, and also by introducing dystrophin mutation into human embryonic stem cells via CRISPR/Cas9. While dystrophin is expressed in healthy hPSC, its deficiency in DMD hPSC lines induces the release of reactive oxygen species (ROS) through dysregulated activity of all three isoforms of nitric oxide synthase (further abrev. as, NOS). NOS-induced ROS release leads to DNA damage and genomic instability in DMD hPSC. We were able to reduce both the ROS release as well as DNA damage to the level of wild-type hPSC by inhibiting NOS activity.

Water Exchange Produces Significantly Higher Adenoma Detection Rate Than Water Immersion: Pooled Data From 2 Multisite Randomized Controlled Trials.

GOALS: To test the hypothesis that water exchange (WE) significantly increases adenoma detection rates (ADR) compared with water immersion (WI). BACKGROUND: Low ADR was linked to increased risk for interval colorectal cancers and related deaths. Two recent randomized controlled trials of head-to-head comparison of WE, WI, and traditional air insufflation (AI) each showed that WE achieved significantly higher ADR than AI, but not WI. The data were pooled from these 2 studies to test the above hypothesis. STUDY: Two trials (5 sites, 14 colonoscopists) that randomized 1875 patients 1:1:1 to AI, WI, or WE were pooled and analyzed with ADR as the primary outcome. RESULTS: The ADR of AI (39.5%) and WI (42.4%) were comparable, significantly lower than that of WE (49.6%) (vs. AI P=0.001; vs. WI P=0.033). WE insertion time was 3 minutes longer than that of AI (P<0.001). WE showed significantly higher detection rate (vs. AI) of the >10 mm advanced adenomas. Right colon combined advanced and sessile serrated ADR of AI (3.4%) and WI (5%) were comparable and were significantly lower than that of WE (8.5%) (vs. AI P<0.001; vs. WI P=0.039). CONCLUSIONS: Compared with AI and WI, the superior ADR of WE offsets the drawback of a significantly longer insertion time. For quality improvement focused on increasing adenoma detection, WE is preferred over WI. The hypothesis that WE could lower the risk of interval colorectal cancers and related deaths should be tested.

Digital cholangioscopy: the diagnostic yield and impact on management of patients with biliary stricture.

OBJECTIVES: Biliary strictures frequently present a diagnostic challenge. The aim of this study was to evaluate the impact of digital single-operator cholangioscopy (DSOC) on subsequent treatment of patients with biliary stricture. METHODS: Consecutive patients undergoing DSOC for biliary stricture were enrolled. Gold standard for final diagnosis included histology from surgical resection. In patients without surgery, clinical evaluation methods and repeated imaging studies were used for comparison of DSOC findings and final diagnosis. Patients were followed-up prospectively focusing on subsequent treatment. RESULTS: Among 30 enrolled patients, final diagnosis was malignant in 13 (43%) and benign in 17 (57%). The sensitivity and specificity of visual impression in diagnosing malignant stricture were 100% (95% CI: 75 - 100) and 76% (95% CI: 50 - 93), respectively. The sensitivity and specificity for biopsy were 92% (95% CI: 62 - 100) and 100% (95% CI: 78 - 100), respectively. One (3%) case of complicating cholangitis with fatal outcome occurred. Final treatment included surgery in 7 (23%), endoscopy in 18 (60%) and chemotherapy in 3 (10%) of patients. CONCLUSIONS: In this study, favorable operating characteristics of DSOC were confirmed. Absolute negative predictive value of visual impression provided reassurance to patients with benign strictures who avoided unnecessary surgery in 53%. One (3%) case of cholangitis with fatal outcome occurred.