Base editor scanning charts the DNMT3A activity landscape.

DNA methylation is critical for regulating gene expression, necessitating its accurate placement by enzymes such as the DNA methyltransferase DNMT3A. Dysregulation of this process is known to cause aberrant development and oncogenesis, yet how DNMT3A is regulated holistically by its three domains remains challenging to study. Here, we integrate base editing with a DNA methylation reporter to perform in situ mutational scanning of DNMT3A in cells. We identify mutations throughout the protein that perturb function, including ones at an interdomain interface that block allosteric activation. Unexpectedly, we also find mutations in the PWWP domain, a histone reader, that modulate enzyme activity despite preserving histone recognition and protein stability. These effects arise from altered PWWP domain DNA affinity, which we show is a noncanonical function required for full activity in cells. Our findings highlight mechanisms of interdomain crosstalk and demonstrate a generalizable strategy to probe sequence-activity relationships of nonessential chromatin regulators.

Validation testing to determine the sensitivity of lateral flow testing for asymptomatic SARS-CoV-2 detection in low prevalence settings: Testing frequency and public health messaging is key.

Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

The Victorian Institute of Sport Assessment Scale for Patellar Tendinopathy (VISA-P): A Reliability Generalization Meta-analysis.

OBJECTIVE: The Victorian Institute of Sport Assessment-Patella (VISA-P) is a questionnaire to assess the severity of patellar tendinopathies. Its use requires good reliability indicators: internal consistency, test-retest and parallel forms. Several studies have been published examining this question, but to date the reliability of this questionnaire (meta-analysis) has not been generalized. The aim of this study was to perform a meta-analysis to generalize the reliability of the VISA-P. DATA SOURCES: MEDLINE, EMBASE, and Scopus. STUDY SELECTION: Studies included were those examining the reliability coefficients of the VISA-P: Cronbach alpha, intraclass correlation coefficient (ICC), and parallel-forms (correlation coefficients compared with other scales). DATA EXTRACTION: All coefficients were extracted and the mean reliability was obtained using fixed- or random-effects models. Sensitivity (leave-one-out analysis) was analyzed. Quality assessment was performed using the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) checklist. DATA SYNTHESIS: Of 364 scientific articles, 12 fulfilled meta-analysis criteria. The summary statistic was 0.86 [95% confidence interval (CI): 0.78-0.92] for Cronbach alpha and 0.94 (95% CI: 0.89-0.97) for the ICC. Parallel forms depended on the comparative test used, ranging from -0.83 to 0.68. The sensitivity analysis found an influential study for the parallel-forms reliability in the Blazina score. We were unable to analyze the asymmetry of funnel plots and meta-regression models because of the number of studies. CONCLUSIONS: The reliability of VISA-P for assessing the severity of patellar tendinopathies requires greater evaluation with more scientific evidence before it can be implemented in clinical practice.

A general presentation on how to carry out a CHARMS analysis for prognostic multivariate models.

The CHARMS (critical appraisal and data extraction for systematic reviews of prediction modelling studies) checklist was created to provide methodological appraisals of predictive models, based on the best available scientific evidence and through systematic reviews. Our purpose is to give a general presentation on how to carry out a CHARMS analysis for prognostic multivariate models, making clear what the steps are and how they are applied individually to the studies included in the systematic review. This tutorial is aimed at providing such a resource. In addition to this explanation, we will apply the method to a real case: predictive models of atrial fibrillation in the community. This methodology could be applied to other predictive models using the steps provided in our review so as to have complete information for each included model and determine whether it can be implemented in daily clinical practice.

Clusters of Cardiovascular Risk Factors and Their Impact on the 20-Year Cardiovascular Risk in a General Population.

BACKGROUND: Clustering of cardiovascular risk factors (CVRFs) is extraordinarily common and is associated with an increased risk of cardiovascular disease (CVD). However, the particular impact of the sum of CVRFs on cardiovascular morbidity and mortality has not been sufficiently explored in Europe. OBJECTIVE: The aim of this study was to analyze the differences in survival-free probability of CVD in relation to the number of CVRFs in a Spanish population. METHODS: A prospective cohort study was conducted from 1992 to 2016 in a Spanish population that included 1144 subjects with no history of CVD (mean age, 46.7 years) drawn from the general population. We calculated the number of CVRFs for each subject (male sex, smoking, diabetes, hypertension, dyslipidemia, obesity, and left ventricular hypertrophy). Cardiovascular morbidity and mortality records were collected, and survival analysis was applied (competing risk models). RESULTS: There were 196 cardiovascular events (17.1%). The differences in total survival-free probability of cardiovascular morbidity and mortality of the different values of the sum of CVRFs were significant, increasing the risk of CVD (hazard ratio, 1.30; 95% confidence interval, 1.13-1.50) per each additional risk factor. CONCLUSION: Differences in survival-free probability of CVD in relation to the number of CVRFs present were statistically significant. Further studies are needed to corroborate our results.

Initiation of Protein Synthesis with Non-Canonical Amino Acids In†Vivo.

By transplanting identity elements into E. coli tRNAfMet , we have engineered an orthogonal initiator tRNA (itRNATy2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

Ultrasound Surveillance in Endovascular Revascularization of Lower Limbs.

BACKGROUND: Endovascular surgery has become the initial treatment for most patients with chronic ischemia of the lower limbs. Few studies support ultrasound surveillance (US) of this kind of procedures. The purpose of this study was to evaluate the initial efficacy of duplex ultrasound as a surveillance method in endovascular treatment in symptomatic peripheral arterial disease patients in our center. MATERIAL AND METHODS: A total of 113 endovascular procedures performed in 106 patients between February 2013 and June 2015 were included. Follow-up included clinical assessment, physical examination, ankle-brachial index (ABI), plethysmography, and ultrasound at 1, 3, 6, 12, 18, and 24 months after surgery. Patients without a minimum follow-up of two controls were excluded. Worsening was defined as follows: (1) in ultrasound, a restenosis >70%; (2) from ABI, a decrease >0.15; (3) clinically, a decrease in claudication distance, reappearance rest pain, or worsening injuries; (4) in plethysmography, flattening in the curve. RESULTS: The average age was 68.3 years, with 72% being men. Twenty-two percent of treated lesions were iliac, 57% were femoropopliteal, and 21% were distal. There were 329 visits, with a mean follow-up of 13.5 months (3-31). The US detected permeability or moderate stenosis in 66 patients (58.4%) and restenosis or occlusion in 47 (41.6%). When compared with clinical status, there was a noncorrelation in 23% and a discrepancy with respect to the ABI of 27% and of 39% with plethysmography. All these differences were statistically significant (P < 0.001). Twenty-one reinterventions were performed (18.6%), six patients died (5.3%), and 11 required major amputation (9.7%). CONCLUSIONS: Clinical status and hemodynamics can detect restenosis or occlusion of the procedure in a large part of the cases, but it can omit more than 20% of these that were only detected by US. The ultrasound follow-up is of great help to increase the reliability of the control in patients with endovascular revascularization of lower limbs.

A systematic review of predictive models for recurrence and mortality in patients with tongue cancer.

INTRODUCTION: Predictive models must meet clinical/methodological standards to be used in clinical practice. However, no critique of those models relating to mortality/recurrence in tongue cancer has been done bearing in mind the accepted standards. METHODS: We conducted a systematic review evaluating the methodology and clinical applicability of predictive models for mortality/recurrence in tongue cancer published in MEDLINE and Scopus. For each model, we analysed (domains of CHARMS, Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modelling Studies) the following: source of data, participants, outcome to be predicted, candidate predictors, sample size, missing data, model development, model performance, model evaluation, results and interpretation and discussion. RESULTS: We found two papers that included eight prediction models, neither of which adhered to the CHARMS recommendations. CONCLUSION: Given the quality of tongue cancer models, new studies following current consensus are needed to develop predictive tools applicable in clinical practice.

Contrast-Induced Nephropathy: A Fact or Fiction in Lower Limb Revascularization?

INTRODUCTION: Contrast-induced nephropathy (CIN) is defined as an increase >25% of serum creatinine from baseline, occurring in 24-48 hours after exposure to contrast, while alternative explanations for renal impairment have been excluded. The volume administered directly relates to risk, increasing by 12% per 100 mL of contrast. According to the series, its incidence varies between 3.3% and 8% in patients without renal damage and 12-50% in patients with chronic kidney disease (CKD) and/or diabetes mellitus (DM). The purpose of this study is to determine the incidence of CIN in endovascular revascularization of lower limbs in our center, where we apply the ALARA concept (As Low As Reasonably Achievable) to the use of contrast. MATERIAL AND METHODS: 163 patients who underwent endovascular revascularization procedures in lower limbs were included in this prospective observational study between February 2013 and April 2015. They were classified according to clinical stage and presence of DM and/or CKD. Data included serum creatinine values preoperative and postoperative, type and volume of contrast used. Patients on hemodialysis and those without sufficient analytical data were excluded. Chi-squared test and Student t-test were used for data analysis. P < 0.05 was considered statistically significant. RESULTS: 109 patients were enrolled, with 67% of DM and 31.5% of CKD. CIN incidence was 3.7% in patients without DM neither CKD, in DM was 6.8% and 12.5% in CKD. Mean creatinine presurgery was 97.96 and postsurgery 97.07, finding no significant differences between them (P = 0.753). Medium-contrast volume was 37.43 mL ± 22.3. The worsening variable (creatinine postsurgery minus creatinine presurgery) was evaluated according to clinical stage, DM, or CKD, being not significant in either group. CONCLUSIONS: In our experience, the dose administered of contrast was not related to the existence of postprocedure CIN, due to the policy of optimizing the use of contrast.

Mandibular cortical indices on cone-beam computed tomography images in osteoporotic women on treatment with oral bisphosphonates.

OBJECTIVE: To evaluate the mandibular cortical indices in osteoporotic women receiving oral bisphosphonates (oBPs). MATERIAL AND METHODS: The study group included 46 Caucasian women aged over 55 years on treatment with oBPs for postmenopausal osteoporosis. Information recorded included age, weight, height, type of oBP, duration of treatment, comorbid conditions and coadjuvant medication. Forty-six age-matched Caucasian women with no known diagnosis of osteoporosis were selected as the control group. All participants underwent cone-beam computed tomography, and the mandibular cortical width (MCW) and the height from the inferior mandibular border to the mental foramen (MBMF) were measured. RESULTS: The MBMF was similar in the participants with osteoporosis and in controls. MCW was significantly greater in the participants with osteoporosis than in the controls (p < 0.001). CONCLUSION: Mandibular cortical width should not be used to predict the risk of bisphosphonate-associated osteonecrosis of the jaws.

Discrepancy in medications reported by elderly patients in the dental office and in their electronic medical records: A pilot study.

AIMS: This study's main objective was to analyze the discrepancy between the dental medication record (DMR) and the physician-prescribed active medications recorded in the medical medication record (MMR). METHODS: The study group consisted of 100 adults who attended the University Dental Clinic (Santiago de Compostela, Spain) requesting dental care. A dental history was created for all participants that included the DMR. The MMR were compiled from their electronic medical records. RESULTS: About 80% of the patients consumed at least one drug (94.2% of those >65 years) and 19% took more than five drugs (26.4% of those > 65 years). In total, 54% of the patients had some discrepancy between the medications recorded in the DMR and those in the MMR (48.4% for those ≤65 years and 64.7% for those >65 years). The rate of participants who omitted some drugs was higher for those >65 years. The drugs most omitted from the DMR were analgesics/opioids, antihypertensives and anxiolytics/hypnotics/sedatives. CONCLUSIONS: It is imperative to access the MMR of patients requesting dental care because a significant number of medications are not reflected in their DMR. These discrepancies may be particularly common and relevant in elderly patients, in whom multimorbidity and polypharmacy are more frequent.

Base Editor Scanning Reveals Activating Mutations of DNMT3A.

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis and acute myeloid leukemia. Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.

Activity-based CRISPR scanning uncovers allostery in DNA methylation maintenance machinery.

Allostery enables dynamic control of protein function. A paradigmatic example is the tightly orchestrated process of DNA methylation maintenance. Despite the fundamental importance of allosteric sites, their identification remains highly challenging. Here, we perform CRISPR scanning on the essential maintenance methylation machinery-DNMT1 and its partner UHRF1-with the activity-based inhibitor decitabine to uncover allosteric mechanisms regulating DNMT1. In contrast to non-covalent DNMT1 inhibition, activity-based selection implicates numerous regions outside the catalytic domain in DNMT1 function. Through computational analyses, we identify putative mutational hotspots in DNMT1 distal from the active site that encompass mutations spanning a multi-domain autoinhibitory interface and the uncharacterized BAH2 domain. We biochemically characterize these mutations as gain-of-function, exhibiting increased DNMT1 activity. Extrapolating our analysis to UHRF1, we discern putative gain-of-function mutations in multiple domains, including key residues across the autoinhibitory TTD-PBR interface. Collectively, our study highlights the utility of activity-based CRISPR scanning for nominating candidate allosteric sites, and more broadly, introduces new analytical tools that further refine the CRISPR scanning framework.

Haploidentical bone marrow transplantation in Mexico.

Haploidentical hematopoietic cell transplantation using CD34(+) cells depleted of T lymphocytes by the CliniMACS is a treatment for hematological malignancy. We report on four Mexican children, three with acute lymphocytic leukemia and one with chronic myelocytic leukemia, who was transplanted with 12 × 10(6) CD34(+) stem cells/kg body weight (98% of purity) with a follow-up of 9½ years. The engraftment was successful in three of the four children. All showed cytomegalovirus reactivation, and one died because of graft rejection and infectious complication. The risk of infections was a major problem.

Criteria for the prescription of oral bisphosphonates for the treatment of osteoporosis in a series of women referred for tooth extraction.

OBJECTIVE: To evaluate the criteria for the prescription of oral bisphosphonates (OB) in a series of women with osteoporosis referred for tooth extraction. STUDY DESIGN: The study included 38 postmenopausal women on treatment with OBs. The following variables were analysed: age, weight, height, type of OB and duration of treatment, bone densitometry and risk factors for osteoporosis. In addition, the osteoporosis self-assessment tool (OST) was administered and collagen type I C-telopeptide (CTX) levels were measured. RESULTS: Bone densitometry had only been performed in six patients (15.7%) before starting OB treatment. Based on the results of the OST, nine (23.6%) of the participants presented a low risk of osteoporosis. CTX levels were measured in 23 patients: 11 (47.8%) presented values below 150 pg/ml. CONCLUSION: Although all patients in the present series were on treatment with OBs, a large percentage did not satisfy the criteria for the initiation of treatment for postmenopausal osteoporosis.

A practical approach to dental care for patients with Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome is a polymalformative chromosomal disorder caused by a deletion in the distal region of the short arm of chromosome 4. The disease is considered rare (1/50,000 births) and predominantly affects females (2:1). In addition to the characteristic facial phenotype ("Greek warrior helmet"), its clinical manifestations include epilepsy, developmental and psychomotor delay, intellectual disability, cardiac and respiratory complications, and eating problems. The most prevalent oral manifestations are hypodontia, delayed tooth eruption, morphological dental abnormalities, dental malocclusions, cleft lip/palate and ogival palate. Based on our clinical experience, Wolf-Hirschhorn syndrome does not represent an absolute contraindication for any type of dental procedure. The feasibility of dental treatment will depend mainly on the degree of epilepsy control and on the level of collaboration, this latter conditioned by the severity of the intellectual disability and communication difficulties.

Usefulness of ultrasound-guided tibio-pedal access in patients with critical limb ischemia.

INTRODUCTION: Retrograde access performed guided by fluoroscopy or ultrasound. We aimed to analyze the usefulness of ultrasound in retrograde access in patients with critical limb ischemia. METHODS: Observational analytical study. From December 2013 to June 2019. We included all retrograde accesses that were guided by ultrasound. Our register assesses demographic and clinical data, the vessel used as retrograde access, the procedure performed, the amount of contrast agent used and time of fluoroscopy, access failure, and local complications. RESULTS: On 715 procedures performed, was used ultrasound-guided retrograde access in 25 patients (64% men). The mean age was 74.8 years (45-90), with 92% of diabetics and 32% of chronic renal failure. Two patients with Rutherford stage 4 and 23 with stage 5-6. In 24 (96%) patients the ultrasound-guided puncture was successful, while in one (4%) of them, it was not possible to enter the target vessel. After the punch, was achieved the technical success of revascularization in 19 (79.2%) patients, with 5 (20.8%) in whom did not the arterial injury was not overcome. The arteries used as retrograde access were: anterior tibial 11, posterior tibial 10, and peroneal in 4. The mean of contrast used was 63 mL (9-100 mL) with an average time of 43 min (15-76 min). Complications related did not observe in retrograde access. CONCLUSIONS: Ultrasound-guided retrograde distal access is an effective method that may use as a bailout method in those endovascular procedures in which it is not possible to cross the lesion anterogradely.

Longitudinal Characterization of a Neutralizing and Total Antibody Response in Patients with Severe COVID-19 and Fatal Outcomes.

The host immune response to SARS-CoV-2 appears to play a critical role in disease pathogenesis and clinical manifestations in severe COVID-19 cases. Until now, the importance of developing a neutralizing antibody response in the acute phase and its relationship with progression to severe disease or fatal outcome among hospitalized patients remains unclear. In this study, we aim to characterize and compare longitudinally the primary humoral immune host response in the early stages of the disease, looking for an association between neutralization, antibody titers, infective viral lineage, and the clinical outcome in hospitalized and non-hospitalized patients. A total of 111 patients admitted at INER from November 2021 to June 2022 were included. We found that patients with negative or low neutralization showed a significant reduction in survival probability compared to patients with medium or high neutralization. We observed a significant decrease in the median of neutralization in patients infected with viral variants with changes in RBD of the spike protein. Our results suggest that developing an early and robust neutralizing response against SARS-CoV-2 may increase survival probability in critical patients.

Surgical treatment of mixed urinary incontinence: effect of anterior colpoplasty.

INTRODUCTION AND HYPOTHESIS: The aims of this study are to study the surgical outcomes in patients with stress-predominant mixed urinary incontinence (MUI) treated with tension-free vaginal tape, and to evaluate the effect of concomitant anterior colpoplasty. METHODS: A total of 134 women with MUI clinical and urodynamic were assessed at 6 months and annually (maximum 9 years) after surgery. RESULTS: As a whole, a complete healing of 49-51% at 3 years is observed, which hence could result in a tax drop of 36.8% at 5-9 years. Nevertheless, the rate of healing or improvement (subjective) is 73.7% at long term (5-9 years). There were no significant differences between the two techniques used: TVT vs TVT-Obturator. On the other hand, we have found significant differences between association of anterior colpoplasty and not (complete healing rate 34.6% vs 64.6%, p = 0.014), at 2 years of monitoring. CONCLUSIONS: MUI with cystocele presents better surgical results for two components of incontinence, with its physiopathogenic implications.