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Background: Random pattern flap is a common reconstructive surgery procedure but its necrosis is a challenging problem. A lot of pharmacological agents and surgical procedures have been examined for the prevention of this complication to maximize the length to width ratio of these surgical flaps. Therefore, we designed an experimental study to evaluate the effects of aspirin, clopidogrel bisulfate (Plavix) and their combination on random skin flap survival in rats.Methods: Forty male rats were randomly assigned to four equal groups. Surgery was done under general anesthesia. A random, rectangular 3×11 cm dorsal skin flap was designed, elevated and sutured back into its primary site. In group one, 100 mg/kg Aspirin and in group two, 25 mg/kg Plavix were administered orally for 7 days postoperatively. Aspirin and Plavix were co-administered in the third group for the same period of time while the control group received no medication. After 7 days, the total surface of flaps, the viable and also the necrotic parts were measured by Image J software. Mean standard deviation and analysis of variance were calculated to compare the results.Results: The mean area of flap survival was 62.49% in the control, 64.04% in Aspirin, 65.09% in Plavix and 64.06% in combination groups. No statistically significant differences were found between treatment groups and control rats.Conclusion: In this study, we found no significant differences between Aspirin, Plavix or their combination on the survival of random skin flaps.

Background:  Telemetry monitoring is crucial for high-risk patients but excessive use beyond practice standards increases costs. Prior studies have shown that electronic health record (EHR) alerts reduce low-value telemetry monitoring. However, specific components of these alerts that contribute to effectiveness are unknown., Objectives:  We aimed to revise previously implemented EHR Best Practice Advisories (BPAs) to optimize their effectiveness in reducing telemetry duration. The secondary objective was to assess the impact on clinicians' alert burden., Methods:  A multicenter retrospective study was conducted at Johns Hopkins Hospital (JHH), Johns Hopkins Bayview Medical Center (JHBMC), and Howard County General Hospital (HCGH). An EHR alert in the form of a BPA was previously implemented at JHH/JHBMC, firing at 24, 48, or 72 hours based on order indication. HCGH used an alert firing every 24 hours. A revised BPA was implemented at all hospitals optimizing the prior JHH/JHBMC alert by including patient-specific telemetry indications, restricting alerts to daytime hours (8:00 a.m.-6:00 p.m.), and embedding the discontinuation order within the BPA alert. A retrospective analysis from October 2018 to December 2021 was performed. The primary outcome was telemetry duration. The secondary outcome was the mean monthly BPA alerts per patient-day., Results:  Compared with the original BPA, the revised BPA reduced telemetry duration by a mean of 6.7 hours (95% CI: 5.2-9.1 hours, p  < 0.001) at JHH/JHBMC, with a minimal increase of 0.06 mean monthly BPA alerts per patient-day ( p  < 0.001). The BPA acceptance rate increased from 7.8 to 31.3% postintervention at JHH/JHBMC ( p  < 0.0001). At HCGH, the intervention led to a mean monthly reduction of 20.2 hours in telemetry duration per hospitalization (95% CI: 19.1-22.8 hours, p  < 0.0001)., Conclusion:  Optimizing EHR BPAs reduces unnecessary telemetry duration without substantially increasing clinician alert burden. This study highlights the importance of tailoring EHR alerts to enhance effectiveness and promote value-based care., Competing Interests: None declared., (Thieme. All rights reserved.)

Mouse models of congenital aortic valve malformations are useful for studying disease pathobiology, but most models have incomplete penetrance [e.g., ∼2 to 77% prevalence of bicuspid aortic valves (BAVs) across multiple models]. For longitudinal studies of pathologies associated with BAVs and other congenital valve malformations, which manifest over months in mice, it is operationally inefficient, economically burdensome, and ethically challenging to enroll large numbers of mice in studies without first identifying those with valvular abnormalities. To address this need, we established and validated a novel in vivo high-frequency (30 MHz) ultrasound imaging protocol capable of detecting aortic valvular malformations in juvenile mice. Fifty natriuretic peptide receptor 2 heterozygous mice on a low-density lipoprotein receptor-deficient background (Npr2 +/- ; Ldlr -/- ; 32 males and 18 females) were imaged at 4 and 8 wk of age. Fourteen percent of the Npr2 +/- ; Ldlr -/- mice exhibited features associated with aortic valve malformations, including 1 ) abnormal transaortic flow patterns on color Doppler (recirculation and regurgitation), 2 ) peak systolic flow velocities distal to the aortic valves reaching or surpassing ∼1,250 mm/s by pulsed-wave Doppler, and 3 ) putative fusion of cusps along commissures and abnormal movement elucidated by two-dimensional (2-D) imaging with ultrahigh temporal resolution. Valves with these features were confirmed by ex vivo gross anatomy and histological visualization to have thickened cusps, partial fusions, or Sievers type-0 bicuspid valves. This ultrasound imaging protocol will enable efficient, cost effective, and humane implementation of studies of congenital aortic valvular abnormalities and associated pathologies in a wide range of mouse models. NEW & NOTEWORTHY We developed a high-frequency ultrasound imaging protocol for diagnosing congenital aortic valve structural abnormalities in 4-wk-old mice. Our protocol defines specific criteria to distinguish mice with abnormal aortic valves from those with normal tricuspid valves using color Doppler, pulsed-wave Doppler, and two-dimensional (2-D) imaging with ultrahigh temporal resolution. This approach enables early identification of valvular abnormalities for efficient and ethical experimental design of longitudinal studies of congenital valve diseases and associated pathologies in mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma that contributes to aggressive tumor biology and therapeutic resistance. Current in vitro PDAC models lack sufficient optical and physical access for fibrous network visualization, in situ mechanical stiffness measurement, and metabolomic profiling. Here, we describe an openable multilayer microfluidic PDAC-on-a-chip platform that consists of pancreatic tumor cells (PTCs) and pancreatic stellate cells (PSCs) embedded in a 3D collagen matrix that mimics the stroma. Our system allows fibrous network visualization via reflected light confocal (RLC) microscopy, in situ mechanical stiffness testing using atomic force microscopy (AFM), and compartmentalized hydrogel extraction for PSC metabolomic profiling via mass spectrometry (MS) analysis. In comparing cocultures of gel-embedded PSCs and PTCs with PSC-only monocultures, RLC microscopy identified a significant decrease in pore size and corresponding increase in fiber density. In situ AFM indicated significant increases in stiffness, and hallmark characteristics of PSC activation were observed using fluorescence microscopy. PSCs in coculture also demonstrated localized fiber alignment and densification as well as increased collagen production. Finally, an untargeted MS study putatively identified metabolic contributions consistent with in vivo PDAC studies. Taken together, this platform can potentially advance our understanding of tumor-stromal interactions toward the discovery of novel therapies.

A significant portion of human cancers are caused by oncoviruses (12%-25%). Oncoviruses employ various strategies to promote their replication and induce tumourigenesis in host cells, one of which involves modifying the gene expression patterns of the host cells, leading to the rewiring of genes and resulting in significant changes in cellular processes and signalling pathways. In recent studies, a specific mode of gene regulation known as circular RNA (circRNA)-mediated competing endogenous RNA (ceRNA) networks has emerged as a key player in this context. CircRNAs, a class of non-coding RNA molecules, can interact with other RNA molecules, such as mRNAs and microRNAs (miRNAs), through a process known as ceRNA crosstalk. This interaction occurs when circRNAs, acting as sponges, sequester miRNAs, thereby preventing them from binding to their target mRNAs and modulating their expression. By rewiring the host cell genome, oncoviruses have the ability to manipulate the expression and activity of circRNAs, thereby influencing the ceRNA networks that can profoundly impact cellular processes such as cell proliferation, differentiation, apoptosis, and immune responses. This review focuses on a comprehensive evaluation of the latest findings on the involvement of virus-induced reprogramming of host circRNA-mediated ceRNA networks in the development and pathophysiology of human viral cancers, including cervical cancer, gastric cancer, nasopharyngeal carcinoma, Kaposi's sarcoma, hepatocellular carcinoma, and diffuse large B cell lymphoma. Understanding these mechanisms can improve our knowledge of how oncoviruses contribute to human tumourigenesis and identify potential targets for developing optimised therapies and diagnostic tools for viral cancers., (© 2024 John Wiley & Sons Ltd.)

Exosomes are a type of extracellular vesicle that contains bioactive molecules that can be secreted by most cells. Nevertheless, the content of these cells differs depending on the cell from which they originate. The exosome plays a crucial role in modulating intercellular communication by conveying molecular messages to neighboring or distant cells. Cancer-derived exosomes can transfer several types of molecules into the tumor microenvironment, including high levels of microRNA (miRNA). These miRNAs significantly affect cell proliferation, angiogenesis, apoptosis resistance, metastasis, and immune evasion. Increasing evidence indicates that exosomal miRNAs (exomiRs) are crucial to regulating cancer resistance to apoptosis. In cancer cells, exomiRs orchestrate communication channels between them and their surrounding microenvironment, modulating gene expression and controlling apoptosis signaling pathways. This review presents an outline of present-day knowledge of the mechanisms that affect target cells and drive cancer resistance to apoptosis. Also, our study looks at the regulatory role of exomiRs in mediating intercellular communication between tumor cells and surrounding microenvironmental cells, specifically stromal and immune cells, to evade therapy-induced apoptosis., Competing Interests: The authors declare that they have no competing interests., (© 2024 The Authors.)

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 μN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.

Objectives: The objective of this study was to evaluate the efficacy of immediate injection treatments of dexamethasone, hyaluronic acid (HA)/gelatin (Ge) hydrogel and glycol-chitosan solution on the phonatory function of rabbit larynges at 42 days after surgical injury of the vocal folds, piloting a novel ex vivo phonatory functional analysis protocol., Methods: A modified microflap procedure was performed on the left vocal fold of 12 rabbits to induce an acute injury. Animals were randomized into one of four treatment groups with 0.1 mL injections of dexamethasone, HA/Ge hydrogel, glycol-chitosan or saline as control. The left mid vocal fold lamina propria was injected immediately following injury. The right vocal fold served as an uninjured control. Larynges were harvested at Day 42 after injection, then were subjected to airflow-bench evaluation. Acoustic, aerodynamic and laryngeal high-speed videoendoscopy (HSV) analyses were performed. HSV segments of the vibrating vocal folds were rated by three expert laryngologists. Six parameters related to vocal fold vibratory characteristics were evaluated on a Likert scale., Results: The fundamental frequency, one possible surrogate of vocal fold stiffness and scarring, was lower in the dexamethasone and HA/Ge hydrogel treatment groups compared to that of the saline control (411.52±11.63 Hz). The lowest fundamental frequency value was observed in the dexamethasone group (348.79±14.99 Hz). Expert visual ratings of the HSV segments indicated an overall positive outcome in the dexamethasone treatment group, though the impacts were below statistical significance., Conclusion: Dexamethasone injections might be used as an adjunctive option for iatrogenic vocal fold scarring. An increased sample size, histological correlate, and experimental method improvements will be needed to confirm this finding. Results suggested a promising use of HSV and acoustic analysis techniques to identify and monitor post-surgical vocal fold repair and scarring, providing a useful tool for future studies of vocal fold scar treatments., (Copyright © 2021 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Background/aim: To identify correlations between urodynamic study (UDS) findings and urinary symptoms in children with refractory monosymptomatic and nonmonosymptomatic primary nocturnal enuresis., Materials and Methods: A total of 96 neurologically normal children were enrolled, 44 consecutive boys and 51 consecutive girls, aged 5-18 years, of whom 41 (38.8%) had refractory monosymptomatic nocturnal enuresis (MNE) and 55 (61.2%) had refractory non-MNE (NMNE). We assessed the urodynamics of all children to detect any underlying bladder overactivity. A comparative analysis was carried out between the two groups of patients., Results: Detrusor overactivity (DO), low bladder capacity, low compliance, and increased postvoid residual (PVR) were identified in 70 (72.9%), 35 (36.5%), 43, and 76 (79.2%) patients, respectively. The mean bladder compliance was 21.66 ± 14.52 mL/cmH 2 O (2-75 cmH 2 O). Of the NMNE patients, 50 (90.9%) had abnormal urodynamic findings, while 40 (97.5%) had abnormal urodynamic findings in the MNE group. There was a statistically significant relationship between NMNE and both increased PVR and abnormal voiding patterns. Both high PVR and DO were significantly associated with obstructive urinary symptoms. Constipation and history of urinary tract infection (UTI) did not significantly correlate with UDS abnormality ( p  = 1.0 and p  = 0.49, respectively)., Conclusion: There was a high prevalence of bladder function disorders in both refractory MNE and NMNE patients in our study. This included small functional capacity, low bladder compliance, and marked DO. A nocturnal enuresis may be the only presenting symptom, however, it may be associated with bladder overactivity, UTI, and constipation; the UDS findings may aid in guiding the assessment and treatment of children suffering from primary refractory nocturnal enuresis and its association with bladder and bowel symptoms., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Introduction: In spite of the results of previous studies regarding the benefits of ultrasonography for diagnosis of elbow fractures in children, the exact accuracy of this imaging modality is still under debate. Therefore, in this diagnostic systematic review and meta-analysis, we aimed to investigate the accuracy of ultrasonography in this regard., Methods: Two independent reviewers performed systematic search in Web of Science, Embase, PubMed, Cochrane, and Scopus for studies published from inception of these databases to May 2023. Quality assessment of the included studies was performed using Quality Assessment Tool for Diagnostic Accuracy Studies (QUADAS-2). Meta-Disc software version 1.4 and Stata statistical software package version 17.0 were used for statistical analysis., Results: A total of 648 studies with 1000 patients were included in the meta-analysis. The pooled sensitivity and specificity were 0.95 (95% CI: 0.93-0.97) and 0.87 (95% CI: 0.84-0.90), respectively. Pooled positive likelihood ratio (PLR) was 6.71 (95% CI: 3.86-11.67), negative likelihood ratio (NLR) was 0.09 (95% CI: 0.03-0.22), and pooled diagnostic odds ratio (DOR) of ultrasonography in detection of elbow fracture in children was 89.85 (95% CI: 31.56-255.8). The area under the summary receiver operating characteristic (ROC) curve for accuracy of ultrasonography in this regard was 0.93. Egger's and Begg's analyses showed that there is no significant publication bias (P=0.11 and P=0.29, respectively)., Conclusion: Our meta-analysis revealed that ultrasonography is a relatively promising diagnostic imaging modality for identification of elbow fractures in children. However, clinicians employing ultrasonography for diagnosis of elbow fractures should be aware that studies included in this meta-analysis had limitations regarding methodological quality and are subject to risk of bias. Future high-quality studies with standardization of ultrasonography examination protocol are required to thoroughly validate ultrasonography for elbow fractures., Competing Interests: None.

Burn is one of the physically debilitating injuries that can be potentially fatal; therefore, providing appropriate coverage in order to reduce possible mortality risk and accelerate wound healing is mandatory. In this study, collagen/exo-polysaccharide (Col/EPS 1-3%) scaffolds are synthesized from rainbow trout (Oncorhynchus mykiss) skins incorporated with Rhodotorula mucilaginosa sp. GUMS16, respectively, for promoting Grade 3 burn wound healing. Physicochemical characterizations and, consequently, biological properties of the Col/EPS scaffolds are tested. The results show that the presence of EPS does not affect the minimum porosity dimensions, while raising the EPS amount significantly reduces the maximum porosity dimensions. Thermogravimetric analysis (TGA), FTIR, and tensile property results confirm the successful incorporation of the EPS into Col scaffolds. Furthermore,the biological results show that the increasing EPS does not affect Col biodegradability and cell viability, and the use of Col/EPS 1% on rat models displays a faster healing rate. Finally, histopathological examination reveals that the Col/EPS 1% treatment accelerates wound healing, through greater re-epithelialization and dermal remodeling, more abundant fibroblast cells and Col accumulation. These findings suggest that Col/EPS 1% promotes dermal wound healing via antioxidant and anti-inflammatory activities, which can be a potential medical process in the treatment of burn wounds., (© 2023 Wiley-VCH GmbH.)

Site-directed RNA base editing enables the transient and dosable change of genetic information and represents a recent strategy to manipulate cellular processes, paving ways to novel therapeutic modalities. While tools to introduce adenosine-to-inosine changes have been explored quite intensively, the engineering of precise and programmable tools for cytidine-to-uridine editing is somewhat lacking behind. Here we demonstrate that the cytidine deaminase domain evolved from the ADAR2 adenosine deaminase, taken from the RESCUE-S tool, provides very efficient and highly programmable editing when changing the RNA targeting mechanism from Cas13-based to SNAP-tag-based. Optimization of the guide RNA chemistry further allowed to dramatically improve editing yields in the difficult-to-edit 5'-CCN sequence context thus improving the substrate scope of the tool. Regarding editing efficiency, SNAP-CDAR-S outcompeted the RESCUE-S tool clearly on all tested targets, and was highly superior in perturbing the β-catenin pathway. NGS analysis showed similar, moderate global off-target A-to-I and C-to-U editing for both tools., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Diabetic wounds are chronic wounds that are currently affecting many patient's quality of life. These wounds are challenging because of the impaired healing cycle and harsh environment. In this study in situ gelling hydrogels based on oxidized carboxymethyl cellulose (OCMC) and gelatin (Gel) were used to hasten the healing rate due to their ease of application. The suggested system in this work is synthesized from entirely natural renewable biomaterials to not only achieve the best biocompatibility and biodegradability but also to develop a sustainable product. The rheological studies showed that the hydrogel is turned into a gel after about 30 s of the mixing process. Moreover, the hydrogel can absorb about ten times its weight, keeping the wound hydrated. In vitro biological investigations indicated optimal biocompatibility, antibacterial, and antioxidant activity for faster tissue regeneration. This product was tested in vivo on normal rats and diabetic mice models to treat full-thickness incisional wounds. Results showed that the OCMC-Gel hydrogel is able to hasten the healing rate in both non-diabetic and diabetic wounds. Pathological examinations of the regenerated skin tissue revealed that the OCMC-Gel treated groups developed much more than the control group., Competing Interests: Declaration of competing interest We declare that this manuscript is original, has not been published elsewhere partially or fully, and is not currently being considered for publication. We know of no conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcomes., (Copyright © 2023. Published by Elsevier B.V.)

Background: In Flexor Pollicis Longus (FPL) injuries, primary repair with end-to-end suture is the treatment of choice. In cases where primary repair is not possible, tendon transfer or tendon grafting is used, each of which has its strengths and weaknesses. We aimed to investigate the effectiveness of each of the above two methods in patients., Methods: Patients with FPL injury who referred to Hazrat Fatemeh Hospital, Tehran, Iran late in 2020 to 2021, if primary tendon repair was not possible, were randomly repaired with tendon transfer or tendon graft. After the appropriate time, the splint was opened and physiotherapy was performed for the patients. Then, at least three months after the repair, the range of motion of the IP and MP joints of the patients thumb was measured and compared in two groups., Results: Ten patients in the tendon transfer group and 10 patients in the tendon graft group were studied. In the secondary repair of FPL with tendon grafting, the range of motion of both IP and MP joints of the thumb was not significantly different compared to repair with tendon transfer., Conclusion: The findings of this research confirm controversies in this field. In order to obtain more accurate results, it is suggested to carry out a research with a larger number of patients and with strict control over the surgical technique and post-operative care, as well as taking into account the morbidities caused by donor tendon removal and examining the overall satisfaction of the patients., Competing Interests: The authors declare no conflicting interests.

In the last decade, significant progress in tissue engineering, repairing, and replacing organs has been achieved. The design and production of scaffolds for tissue engineering are one of the main areas which have attracted the researcher's interest. In this regard, electrospinning is one of the most popular methods of nanoscale scaffold similar to extracellular matrix production. This paper reports the fabrication of scaffolds consisting of radially aligned PCL nanofibers by utilizing a collector composed of a central point electrode and a peripheral ring electrode. The chemical and physical properties were compared using SEM, FTIR, XRD, and DSC experiments, as well as biological performance using the MTT method and cell morphology with nanofibers with random and unidirectionally morphology. Results of this study showed greater physical and biological properties for radially aligned nanofibers which make them an excellent candidate for wound healing applications due to the guided cell growth on this type of nanofiber., (© TÜBİTAK.)

Background: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) have tremendous promise for application in cardiac regeneration, but their translational potential is limited by an immature phenotype. We hypothesized that large-scale manufacturing of mature hPSC-CMs could be achieved through culture on polydimethylsiloxane (PDMS)-lined roller bottles and that the transplantation of these cells would mediate better structural and functional outcomes than with conventional immature hPSC-CM populations., Methods: We comprehensively phenotyped hPSC-CMs after in vitro maturation for 20 and 40 days on either PDMS or standard tissue culture plastic substrates. All hPSC-CMs were generated from a transgenic hPSC line that stably expressed a voltage-sensitive fluorescent reporter to facilitate in vitro and in vivo electrophysiological studies, and cardiomyocyte populations were also analyzed in vitro by immunocytochemistry, ultrastructure and fluorescent calcium imaging, and bulk and single-cell transcriptomics. We next compared outcomes after the transplantation of these populations into a guinea pig model of myocardial infarction using end points including histology, optical mapping of graft- and host-derived action potentials, echocardiography, and telemetric electrocardiographic monitoring., Results: We demonstrated the economic generation of >1×10 8 mature hPSC-CMs per PDMS-lined roller bottle. Compared with their counterparts generated on tissue culture plastic substrates, PDMS-matured hPSC-CMs exhibited increased cardiac gene expression and more mature structural and functional properties in vitro. More important, intracardiac grafts formed with PDMS-matured myocytes showed greatly enhanced structure and alignment, better host-graft electromechanical integration, less proarrhythmic behavior, and greater beneficial effects on contractile function., Conclusions: We describe practical methods for the scaled generation of mature hPSC-CMs and provide the first evidence that the transplantation of more mature cardiomyocytes yields better outcomes in vivo.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Introduction: This multicentered cohort study evaluated factors associated with patient-centered outcomes of immature permanent teeth that received regenerative endodontic procedures (REPs) or apexification treatment (APEX)., Methods: A record review identified teeth treated with REPs or APEX between September 2005 and December 2014. Data regarding treatment and patient-centered outcomes were extracted from records with a 3-month minimum recall. When possible, participants presented for an in-person prospective research visit. Patient-centered success was defined as an asymptomatic, functional tooth not requiring further endodontic or surgical intervention after completion of the original treatment during the study observation. Risk ratios and adjusted and unadjusted Cox proportional hazard ratios were calculated., Results: The analytic cohort of 187 individuals included 211 teeth (93 REPs and 118 APEX) with an average follow-up of 32 months. Most cases were successful (81% REPs and 92% APEX) and survived the observation period (96% REPs and 97% APEX). The success rate of REPs was lower than APEX and decreased more rapidly over time. Cox regression analysis demonstrated that when controlling for other variables, the association between treatment type and outcome is not significant. Preoperative infection, teeth with more immature roots, and REP treatment are potentially important predictors. Among teeth receiving REPs, a lower failure rate was observed for teeth that received multiantibiotic paste (3/43) compared with calcium hydroxide (11/45)., Conclusions: Teeth receiving REPs required clinical intervention earlier than teeth that received APEX treatment, although a preoperative abscess and more immature root also affected this outcome. Using multiantibiotic paste versus calcium hydroxide in REPs may improve success., (Copyright © 2021. Published by Elsevier Inc.)

The SNAP-ADAR tool enables precise and efficient A-to-I RNA editing in a guideRNA-dependent manner by applying the self-labeling SNAP-tag enzyme to generate RNA-guided editases in cell culture. Here, we extend this platform by combining the SNAP-tagged tool with further effectors steered by the orthogonal HALO-tag. Due to their small size (ca. 2 kb), both effectors are readily integrated into one genomic locus. We demonstrate selective and concurrent recruitment of ADAR1 and ADAR2 deaminase activity for optimal editing with extended substrate scope and moderate global off-target effects. Furthermore, we combine the recruitment of ADAR1 and APOBEC1 deaminase activity to achieve selective and concurrent A-to-I and C-to-U RNA base editing of endogenous transcripts inside living cells, again with moderate global off-target effects. The platform should be readily transferable to further epitranscriptomic writers and erasers to manipulate epitranscriptomic marks in a programmable way with high molecular precision., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many children born with congenital heart disease need a heart valve repair or replacement. Currently available repair materials and valve replacements are incapable of growth, repair, and adaptation , rendering them inadequate for growing children. Heart valve tissue engineering (HVTE) aims to develop living replacement valves that can meet these needs. Among numerous cell sources for in vitro HVTE, umbilical cord perivascular cells (UCPVCs) are particularly attractive because they are autologous, readily available, and have excellent regenerative capacity. As an essential step toward preclinical testing of heart valves engineered from UCPVCs, the goal of this study was to establish methods to isolate, expand, and promote extracellular matrix (ECM) synthesis by UCPVCs from pigs (porcine umbilical cord perivascular cells [pUCPVCs]), as a relevant preclinical model. We determined that Dulbecco's modified Eagle's medium with 20% fetal bovine serum supported isolation and substantial expansion of pUCPVCs, whereas media designed for human mesenchymal stromal cell (MSC) expansion did not. We further demonstrated the capacity of pUCPVCs to synthesize the main ECM components of heart valves (collagen type I, elastin, and glycosaminoglycans), with maximal collagen and elastin per-cell production occurring in serum-free culture conditions using StemMACS™ MSC Expansion Media. Altogether, these results establish protocols that enable the use of pUCPVCs as a viable cell source for preclinical testing of engineered heart valves. Impact statement This study establishes methods to successfully isolate, expand, and promote the synthesis of the main extracellular matrix components of heart valves (collagen type I, elastin, and glycosaminoglycans) by porcine umbilical cord perivascular cells (pUCPVCs). These protocols enable further evaluation of pUCPVCs as an autologous, readily available, and clinically relevant cell source for preclinical testing of pediatric tissue-engineered heart valves.

β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.

Tendon tissue engineering based on stem cell differentiation has attracted a great deal of attention in recent years. Previous studies have examined the effect of cell-imprinted polydimethylsiloxane (PDMS) substrate on induction differentiation in stem cells. In this study, we used tenocyte morphology as a positive mold to create a tenocyte-imprinted substrate on PDMS. The morphology and topography of this tenocyte replica on PDMS was evaluated with scanning electron microscopy (SEM) and atomic force microscopy. The tenogenic differentiation induction capacity of the tenocyte replica in adipose tissue-derived mesenchymal stem cells (ADSCs) was then investigated and compared with other groups, including tissue replica (which was produced similarly to the tenocyte replica and was evaluated by SEM), decellularized tendon, and bone morphogenic protein (BMP)-12, as other potential inducers. This comparison gives us an estimate of the ability of tenocyte-imprinted PDMS (called cell replica in the present study) to induce differentiation compared to other inducers. For this reason, ADSCs were divided into five groups, including control, cell replica, tissue replica, decellularized tendon and BMP-12. ADSCs were seeded on each group separately and investigated by the real-time reverse transcription polymerase chain reaction (RT-PCR) technique after seven and 14 days. Our results showed that in spite of the higher effect of the growth factor on tenogenic differentiation, the cell replica can also induce tenocyte marker expression (scleraxis and tenomodulin) in ADSCs. Moreover, the tenogenic differentiation induction capacity of the cell replica was greater than tissue replica. Immunocytochemistry analysis revealed that ADSCs seeding on the cell replica for 14 days led to scleraxis and tenomodulin expression at the protein level. In addition, immunohistochemistry indicated that contrary to the promising results in vitro, there was little difference between ADSCs cultured on tenocyte-imprinted PDMS and untreated ADSCs. The results of such studies could lead to the production of inexpensive cell culture plates or biomaterials that can induce differentiation in stem cells without growth factors or other supplements.

A matrix derived from natural tissue functions as a highly biocompatible and versatile scaffold for tissue engineering applications. It can act as a supportive construct that provides a niche for colonization by host cells. In this work, we describe a cost-effective, reliable and reproducible protocol for decellularization and preservation of human skin as a potential soft tissue replacement. The decellularized human skin is achieved using purely chemical agents without any enzymatic steps. The suitability of the proposed method for the preservation of the extracellular matrix (ECM) structure and its main components and integrity were evaluated using histological and immunohistochemical analysis. Cryopreservation and final sterility were conducted using programmable freeze-drying and gamma irradiation. The architecture, basement membrane and 3D structure of ECM can be successfully preserved after decellularization. Our protocol was found to be appropriate to maintain key proteins such as collagen type I, III, IV and laminin in the structure of final scaffold. This protocol offers a novel platform for the preparation of a dermal substitute for potential clinical applications. STATEMENT OF SIGNIFICANCE: Clinical application of naturally-based scaffolds for verity of health problems obliges development of a reproducible and effective technology that does not change structural and compositional material properties during scaffold preparation and preservation. Lack of an effective protocol for the production of biological products using decellularization method is still remaining. This effort is directing to solve this challenge in order to accomplish the off-the -shelf availability of decellularized dermal scaffold in market for clinical application., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Human chorionic gonadotropin (hCG) assays are used to detect pregnancy, and urine point-of-care tests are frequently used to triage patients. Under certain conditions, urine tests can fail to detect pregnancy, which can have serious consequences for patient management., Objectives: To understand the prevalence of different factors contributing to false-negative urinary hCG testing results at our institution., Methods: Clinical data for patients with negative urine hCG results and subsequent positive or equivocal serum hCG results within a 1-year period were reviewed., Results: Out of 9447 negative urine hCG results, 11 potential missed diagnoses were identified, with early gestational age as the most common factor, followed by β-core hook effects., Conclusions: Although false-negative urine hCG test results are rare, understanding the commonly encountered reasons for inaccurate testing results can help clinical centers develop strategies to minimize risk for patients., (© American Society for Clinical Pathology 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Porous composite hydrogels were prepared using glycol chitosan as the matrix, glyoxal as the chemical crosslinker, and carbon nanotubes (CNTs) as the fibers. Both carboxylic and hydroxylic functionalized CNTs were used. The homogeneity of CNTs dispersion was evaluated using scanning electron microscopy. Human vocal fold fibroblasts were cultured and encapsulated in the composite hydrogels with different CNT concentrations to quantify cell viability. Rheological tests were performed to determine the gelation time and the storage modulus as a function of CNT concentration. The gelation time tended to decrease for low concentrations and increase at higher concentrations, reaching a local minimum value. The storage modulus obeyed different trends depending on the functional group. The porosity of the hydrogels was found to increase by 120% when higher concentrations of carboxylic CNTs were used. A high porosity may promote cell adhesion, migration, and recruitment from the surrounding native tissue, which will be investigated in a future work aiming at applying this injectable biomaterial for vocal fold tissue regeneration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control., Methods: We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBB IVS1-110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34 + hematopoietic stem cells (HSCs)., Results: Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBB IVS1-110 mutation showed indels in both K562 cells (up to 77%) and CD34 + hematopoietic stem cells-HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34 + HSCs., Conclusion: Our approach provides guidance on non-viral gene correction in CD34 + HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.

Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNA hCFTR in vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNA hCFTR in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNA hCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNA hCFTR was quantified by hCFTR ELISA, and cmRNA hCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV 0.1 , suggesting NP-cmRNA hCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.

Among different graft materials for craniofacial reconstruction, calcium phosphate cements have the advantages of alloplastic grafts and wide use. The authors report a case of foreign body reaction following frontal reconstruction with JectOS (an injectable calcium orthophosphate cement; Kasios) and reviewed the literature on complications of this material after craniofacial reconstruction from 2002 to 2017. Complications were categorized into two groups: immunologic reactions (consisting of seroma collection, chronic sinus mucosa swelling, and foreign body reaction) and non-immune events (infection, fragmentation, and ejection). It is wise to use calcium phosphate-based material only in selected cases with small defects, and long-term follow-up is needed to observe their consequences., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Bottled water is one of the sources of drinking water in many arid and semi-arid countries, including Iran. The greatest concern is the health effects of exposure to excessive nitrate concentrations in drinking water due to the development of methemoglobinemia in children. Therefore, the present study was aimed at determining the concentration of nitrate and its risk assessment in drinking water bottled in Iran. 71 different bottled water brands were identified in this study. The nitrate concentration in water samples was then measured using an Ion Chromatography No. 4110 in accordance Standard Methods for the Examination of Water and Wastewater. The hazard quotient (HQ) of nitrate was calculated using the formula based on input variables including nitrate concentration, water per capita, body weight and reference dose. The results showed that the concentration of nitrate in bottled water was in the range of 0.146-50.1 mg/L (average 10.55 mg/L) in one of which, the concentration of nitrate was higher than the WHO guideline. The mean EDI values for nitrate in different groups of infants, children, teenagers and adults were 0.0795, 0.5633, 0.3976 and 0.3186 mg/kg, respectively. The mean HQ values for nitrate in different groups of infants, children, teenagers and adults were 0.0528, 0.3737, 0.2638 and 0.2114, respectively. In general, the hazard quotient (HQ>1) for the population consuming bottled water, appropriate strategies should be considered in order to reduce the concentration of nitrate in bottled water.

While collagen type I (Col-I) is commonly used as a structural component of biomaterials, collagen type III (Col-III), another fibril forming collagen ubiquitous in many soft tissues, has not previously been used. In the present study, the novel concept of an injectable hydrogel with semi-interpenetrating polymeric networks of heterotypic collagen fibrils, with tissue-specific Col-III to Col-I ratios, in a glycol-chitosan matrix was investigated. Col-III was introduced as a component of the novel hydrogel, inspired by its co-presence with Col-I in many soft tissues, its influence on the Col-I fibrillogenesis in terms of diameter and mechanics, and its established role in regulating scar formation. The hydrogel has a nano-fibrillar porous structure, and is mechanically stable under continuous dynamic stimulation. It was found to provide a longer half-life of about 35 days than similar hyaluronic acid-based hydrogels, and to support cell implantation in terms of viability, metabolic activity, adhesion and migration. The specific case of pure Col-III fibrils in a glycol-chitosan matrix was investigated. The proposed hydrogels meet many essential requirements for soft tissue engineering applications, particularly for mechanically challenged tissues such as vocal folds and heart valves.