FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Human activation-induced deaminase lacks strong replicative strand bias or preference for cytosines in hairpin loops.

Activation-induced deaminase (AID) is a DNA-cytosine deaminase that mediates maturation of antibodies through somatic hypermutation and class-switch recombination. While it causes mutations in immunoglobulin heavy and light chain genes and strand breaks in the switch regions of the immunoglobulin heavy chain gene, it largely avoids causing such damage in the rest of the genome. To help understand targeting by human AID, we expressed it in repair-deficient Escherichia coli and mapped the created uracils in the genomic DNA using uracil pull-down and sequencing, UPD-seq. We found that both AID and the human APOBEC3A preferentially target tRNA genes and transcription start sites, but do not show preference for highly transcribed genes. Unlike A3A, AID did not show a strong replicative strand bias or a preference for hairpin loops. Overlapping uracilation peaks between these enzymes contained binding sites for a protein, FIS, that helps create topological domains in the E. coli genome. To confirm whether these findings were relevant to B cells, we examined mutations from lymphoma and leukemia genomes within AID-preferred sequences. These mutations also lacked replicative strand bias or a hairpin loop preference. We propose here a model for how AID avoids causing mutations in the single-stranded DNA found within replication forks.

What Is the Likelihood of Union and Frequency of Complications After Parallel Plating and Supplemental Bone Grafting for Resistant Distal Femoral Nonunions?

BACKGROUND: Management of resistant distal femur nonunions is challenging because patients not only have disability from an unhealed fracture, but also often have a shortened femur, stiff knee, deformities, and bone defects to address during revision surgery. Dual plating of the distal femur in such a setting can maintain stability that allows the nonunion to heal while also addressing bone defects and correcting deformities simultaneously. Dual-plating techniques that have been described lack standardization with regard to the size and type of medial-side implants and configuration of the dual-plate construct. QUESTIONS/PURPOSES: (1) What proportion of patients achieve radiologic evidence of union after parallel plating of resistant distal femoral nonunions? (2) What improvements in function are achieved with this approach, as assessed by improvements in femoral length discrepancy, knee flexion, and patient-reported outcome scores? (3) What complications are associated with the technique? METHODS: Between 2017 and 2020, the senior author of this study treated 38 patients with resistant distal femoral nonunions, defined here as nonunions that persisted for more than 12 months since the injury despite a minimum of two previous internal fixation procedures. During the study period, our preferred technique for treating aseptic, resistant distal femoral nonunions was to use dual plates in a parallel configuration augmented with autografts. Of 38 patients, three patients with active signs of infection who underwent resection and reconstruction using bone transport techniques and two patients older than 65 years with deficient distal femur bone stock who underwent endoprosthetic reconstruction were excluded. Of the 33 included patients, 67% (22 of 33) were male. The median age was 40 years (range 20 to 67 years). Nonunion was articular and metaphyseal in 13 patients and metaphyseal only in 20 patients. Our surgical approach was to remove existing implants, perform intraoperative culturing to rule out infection, debride the nonunion, correct the deformity, perform intra-articular and extra-articular lysis of adhesions with quadriceps release, and apply fixation using medial and lateral fixed-angle anatomic locked implants positioned in a parallel configuration. Every attempt to improve length was undertaken, and the defects were filled with autografts. A total of 97% of patients were followed until union occurred (one of 33 was lost to follow-up before union was documented), and 79% (26 of 33) were assessed for functional outcomes at a minimum of 2 years (median 38 months [range 25 to 60 months]) after excluding patients lost to follow-up and those in whom union did not occur after parallel plating. Union was defined as evidence of central trabecular bridging on AP radiographs and posterior cortical bridging on lateral radiographs. These radiologic criteria were defined to overcome difficulties in assessing radiologic healing in patients with lateral and medial plates. With parallel plating, bridging trabecular bone along the posterior cortex on lateral radiographs and the central region on AP radiographs is visualized and can be appreciated and interpreted as evidence of healing in two orthogonal planes. Preoperative and follow-up clinical assessment of knee ROM, the extent of femoral length correction based on calibrated femoral radiographs before and after surgery, and the evaluation of improvement in lower limb function based on the preoperative and follow-up differences in responses to the lower extremity functional scale (LEFS) were studied (the LEFS is scored from 0 to 80, with higher scores representing better function). Complications and secondary surgical procedures to address them were abstracted from a longitudinally maintained trauma database. RESULTS: Sixty-seven percent (22 of 33) of nonunions showed radiologic healing by 24 weeks, and another 24% (eight of 33) healed by 36 weeks. Six percent (two of 33) did not unite, and one patient was lost to follow-up before union was documented. In the 79% (26 of 33) of patients available for final functional outcome assessment, the median femoral shortening had improved from 2.4 cm (range 0 to 4 cm) to 1.1 cm (range 0 to 2.3 cm; p < 0.001), and the median knee ROM had improved from 70° (range 20° to 110°) to 100° (range 50° to 130°; p = 0.002) after surgery. The median LEFS score improved to 63 (range 41 to 78) compared with 22 (range 15 to 33; p < 0.001) before surgery. Serious complications, including major thromboembolic events, iliac graft site infection, knee stiffness (flexion < 60°), and medial plate impingement necessitating removal, were seen in 30% (10 of 33) of patients. Secondary surgical interventions were performed in 24% (eight of 33) of patients to address procedure-related complications. CONCLUSION: Based on our findings, a high likelihood of union and improvements in knee and lower limb function can be expected with parallel plating of resistant distal femur nonunions using anatomic locked plates. However, the increased frequency of complications observed in our study suggests the need for improvements in dual-plating techniques and to explore possible alternative fixation methods through larger multicenter comparative studies. LEVEL OF EVIDENCE: Level IV, therapeutic study.

A hybrid thyroid tumor type classification system using feature fusion, multilayer perceptron and bonobo optimization.

BACKGROUND: Thyroid tumor is considered to be a very rare form of cancer. But recent researches and surveys highlight the fact that it is becoming prevalent these days because of various factors. OBJECTIVES: This paper proposes a novel hybrid classification system that is able to identify and classify the above said four different types of thyroid tumors using high end artificial intelligence techniques. The input data set is obtained from Digital Database of Thyroid Ultrasound Images through Kaggle repository and augmented for achieving a better classification performance using data warping mechanisms like flipping, rotation, cropping, scaling, and shifting. METHODS: The input data after augmentation goes through preprocessing with the help of bilateral filter and is contrast enhanced using dynamic histogram equalization. The ultrasound images are then segmented using SegNet algorithm of convolutional neural network. The features needed for thyroid tumor classification are obtained from two different algorithms called CapsuleNet and EfficientNetB2 and both the features are fused together. This process of feature fusion is carried out to heighten the accuracy of classification. RESULTS: A Multilayer Perceptron Classifier is used for classification and Bonobo optimizer is employed for optimizing the results produced. The classification performance of the proposed model is weighted using metrics like accuracy, sensitivity, specificity, F1-score, and Matthew's correlation coefficient. CONCLUSION: It can be observed from the results that the proposed multilayer perceptron based thyroid tumor type classification system works in an efficient manner than the existing classifiers like CANFES, Spatial Fuzzy C means, Deep Belief Networks, Thynet and Generative adversarial network and Long Short-Term memory.

Comparison of ultrasonographic measurement of gastric antral volume and pH with or without pharmacological acid aspiration prophylaxis in low-risk surgical patients - A randomized clinical trial.

Background and Aims: The role of preoperative pharmacological prophylaxis in preventing aspiration pneumonitis under general anesthesia (GA) in patients at low risk of aspiration pneumonitis is still under debate. We addressed the need for routine pharmacological aspiration prophylaxis in at-risk population by assessing the change in gastric volume using ultrasound with and without pharmacological acid aspiration prophylaxis. Material and Methods: A single-center, randomized double-blinded trial, with 200 adult patients scheduled for elective surgical procedures under GA, were randomized into a prophylaxis group, in which the patients received oral famotidine and metoclopramide, and a no prophylaxis group, in which the patients did not receive any prophylaxis. Gastric volume derived from preinduction measurement of gastric antral volume by ultrasound, postinduction gastric pH, and incidences of aspiration pneumonitis were compared. Bland-Altman plot was used to determine the level of agreement between measured gastric volume and ultrasonography based on calculated gastric volume. Results: The gastric antral cross-sectional area (CSA) and volume in the no prophylaxis group (3.12 cm2 and 20.11 ml, respectively) were comparable to the prophylaxis group (2.56 cm2 and 19.67 ml, respectively) (P-values 0.97 and 0.63, respectively). Although there was a statistically significant decrease in gastric pH in the no prophylaxis group (P-value 0.01), it was not clinically significant to increase the risk of aspiration pneumonitis based on Roberts and Shirley criteria (P-value 0.39). Conclusion: In an adequately fasted low-risk population, the amount of residual gastric volume was similar and below the aspiration threshold, regardless of the aspiration prophylaxis status.

Learning from national implementation of the Veterans Affairs Clinical Resource Hub (CRH) program for improving access to care: protocol for a six year evaluation.

BACKGROUND: The Veterans Affairs (VA) Clinical Resource Hub (CRH) program aims to improve patient access to care by implementing time-limited, regionally based primary or mental health staffing support to cover local staffing vacancies. VA's Office of Primary Care (OPC) designed CRH to support more than 1000 geographically disparate VA outpatient sites, many of which are in rural areas, by providing virtual contingency clinical staffing for sites experiencing primary care and mental health staffing deficits. The subsequently funded CRH evaluation, carried out by the VA Primary Care Analytics Team (PCAT), partnered with CRH program leaders and evaluation stakeholders to develop a protocol for a six-year CRH evaluation. The objectives for developing the CRH evaluation protocol were to prospectively: 1) identify the outcomes CRH aimed to achieve, and the key program elements designed to achieve them; 2) specify evaluation designs and data collection approaches for assessing CRH progress and success; and 3) guide the activities of five geographically dispersed evaluation teams. METHODS: The protocol documents a multi-method CRH program evaluation design with qualitative and quantitative elements. The evaluation's overall goal is to assess CRH's return on investment to the VA and Veterans at six years through synthesis of findings on program effectiveness. The evaluation includes both observational and quasi-experimental elements reflecting impacts at the national, regional, outpatient site, and patient levels. The protocol is based on program evaluation theory, implementation science frameworks, literature on contingency staffing, and iterative review and revision by both research and clinical operations partners. DISCUSSION: Health systems increasingly seek to use data to guide management and decision-making for newly implemented clinical programs and policies. Approaches for planning evaluations to accomplish this goal, however, are not well-established. By publishing the protocol, we aim to increase the validity and usefulness of subsequent evaluation findings. We also aim to provide an example of a program evaluation protocol developed within a learning health systems partnership.

Validation of a Persian version of an English language ecologically-valid assessment of executive functions through childhood and adolescence.

Childhood and adolescence are pivotal periods for cognitive development. Executive functions are crucial for efficient cognitive functioning, so accurate assessment is important. One ecologically-valid virtual reality test is the Jansari assessment of Executive Functions for Children (JEF-C©). In a cross-sectional study, we aimed at translating, adapting and validating JEF-C into Persian, and at investigating whether this Persian version (JEF-C (P)) can identify stages of development of executive functions in children aged from 8 to 16. Children and adolescents (N = 146) falling into three age groups participated: 8-10, 11-13 and 14-16 years old. They completed JEF-C (P) and the Wisconsin Card Sorting Test (WCST). There were acceptable Cronbach's alpha coefficients for JEF-C(P) total score (α = .72) and all constructs, except action-based prospective memory, had a positive impact on total internal consistency. There was an effect of age group on overall JEF-C (P) performance and of age on four constructs. There was also a correlation between the number of categories on WCST and the prioritization construct of JEF-C (P). It seems that JEF-C (P) is an ecologically valid executive function assessment sensitive to age and could be useful for both researchers and clinicians working with children.

Transcription-associated mutagenesis.

Transcription requires unwinding complementary DNA strands, generating torsional stress, and sensitizing the exposed single strands to chemical reactions and endogenous damaging agents. In addition, transcription can occur concomitantly with the other major DNA metabolic processes (replication, repair, and recombination), creating opportunities for either cooperation or conflict. Genetic modifications associated with transcription are a global issue in the small genomes of microorganisms in which noncoding sequences are rare. Transcription likewise becomes significant when one considers that most of the human genome is transcriptionally active. In this review, we focus specifically on the mutagenic consequences of transcription. Mechanisms of transcription-associated mutagenesis in microorganisms are discussed, as is the role of transcription in somatic instability of the vertebrate immune system.

Prebiotic potential of enzymatically produced ulvan oligosaccharides using ulvan lyase of Bacillus subtilis, NIOA181, a macroalgae-associated bacteria.

AIMS: To characterize the polysaccharide hydrolyzing potential of macroalgae-associated bacteria (MABs) for the enzymatic production of oligosaccharides and determining their prebiotic potential. METHODS AND RESULTS: Approximately 400 MABs were qualitatively characterized for polysaccharide hydrolyzing activity. Only about 5%-15% of the isolates were found to have the potential for producing porphyranase, alginate lyase and ulvan lyase enzymes, which were quantified in specific substrate broths. One potential MAB, Bacillus subtilis, NIOA181, isolated from green macroalgae, showed the highest ulvan lyase activity. This enzyme was partially purified and used to hydrolyse ulvan into ulvan oligosaccharides. Structural characterization of ulvan oligosaccharides showed that they are predominantly composed of di-, tri- and tetrasaccharide units. Results showed that the enzymatically produced ulvan oligosaccharides exhibited prebiotic activity by promoting the growth of probiotic bacteria and suppressing the enteric pathogens, which were higher than the ulvan polysaccharide and equivalent to commercial fructooligosaccharides. CONCLUSIONS: A potential MAB, NIOA181, producing ulvan lyase was isolated and used for the production of ulvan oligosaccharides with prebiotic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Rarely studied ulvan oligosaccharides with prebiotic activity can be widely used as an active pharmaceutical ingredient in nutraceutical and other healthcare applications.

Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks.

The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.

What Factors Are Associated With Poor Shoulder Function and Serious Complications After Internal Fixation of Three-part and Four-part Proximal Humerus Fracture-dislocations?

BACKGROUND: Three-part and four-part fracture-dislocations of the proximal humerus are characterized by severe soft tissue disruptions that can compromise the viability of the humeral head. As a result, nonunion and avascular necrosis are more common in these injuries. In such injuries, surgical treatment (internal fixation or arthroplasty) is performed in most patients who are determined to be fit for surgery to potentially restore shoulder function. Although the decision to preserve or replace the humeral head is simple in young patients or those > 65 years, in most other patients, the decision can be complicated, and little is known about which patient-related and injury-related factors may be independently associated with poor shoulder function or complications like avascular necrosis. QUESTIONS/PURPOSES: (1) What proportion of fractures united after internal fixation of a three-part or four-part fracture-dislocation of the proximal humerus, what is the mean Constant score at a minimum of 2 years after this injury, and what proportion had serious complications (such as loss of fixation, nonunion, reoperation, or avascular necrosis)? (2) After controlling for potential confounding variables, what factors are independently associated with poor shoulder function (defined as a Constant score < 55 out of 100) and occurrence of serious complications such as loss of fixation or reduction resulting in revision surgery, nonunion, or radiographic evidence of avascular necrosis of the humeral head? METHODS: Between 2011 and 2017, the senior author of this study (ASG) treated 69 patients with three-part or four-part proximal humerus fracture dislocations. During this time, indications for internal fixation in these patients were adequate humeral bone quality as determined by the surgeon on radiographs, adequate bone stock and volume available for fixation in the humeral head as determined on CT images, and the absence of a head split component as assessed on preoperative radiographs and CT images. On this basis, 87% (60 patients) underwent internal fixation with a locked plate and suture fixation of the tuberosities through a deltopectoral approach. Thirteen percent (nine patients) underwent either a hemiarthroplasty or a reverse total shoulder arthroplasty. Of the 60 patients who underwent internal fixation, four declined to participate in the study and two with brachial plexus palsy were not considered for inclusion. This study focused on the remaining 54 patients who were considered potentially eligible. To be included, a minimum follow-up of 2 years was required; 11% (6 of 54) were lost before that time, and the remaining 48 patients were analyzed at a mean of 48 months ± 17 months in this retrospective study, which drew data from longitudinally maintained institutional databases. Fracture union was assessed by obliteration of fracture lines and the presence of bridging trabecular bone on plain radiographs. Shoulder function was assessed using the Constant score, which is scored from 0 to 100 points, with 0 indicating the most disability and 100 the least disability. The anchor-based minimal clinically important difference for the Constant score is 9.8 points. Twelve patient-related and injury-related factors were analyzed using a multivariate regression model to identify factors that are independently associated with poor results after internal fixation as measured by shoulder function and the occurrence of serious complications. We categorized results as poor if patients had one or more of the following: Constant score < 55 out of 100 at the last follow-up examination (for patients who underwent revision surgery, the Constant score immediately before revision was considered) and loss of fixation or reduction resulting in revision surgery, nonunion, or avascular necrosis of the humeral head. Patients were screened for avascular necrosis at 6 and 12 months after surgery, then annually for another 2 years. Further assessments were made only based on symptoms. RESULTS: Seventy-nine percent of the fractures united within 18 weeks of surgery (38 of 48), and an additional 13% united by 24 weeks (6 of 48), while 8% did not unite (4 of 48). The mean Constant score at the last follow-up was 68 ± 12. Twenty-one percent (10 of 48) had a Constant score < 55, indicating poor shoulder function. Twenty-one percent (10 of 48) experienced avascular necrosis, and 15% (7 of 48) with either nonunion or avascular necrosis underwent revision shoulder arthroplasty. Two patients who underwent arthroplasty had both nonunion and avascular necrosis. After controlling for potentially confounding variables, we found that being a woman (odds ratio 1.7 [95% confidence interval 1.4 to 2.1]; p = 0.01), four-part fracture dislocations (OR 2.1 [95% CI 1.5 to 2.7]; p < 0.001), absence of a metaphyseal head extension (OR 2.4 [95% CI 1.8 to 3.3]; p < 0.001), absence of active back-bleeding from the humeral head (OR 3.4 [95% CI 2.3 to 5.1]; p < 0.001), height of the head segment < 2 cm (OR 2.3 [95% CI 1.8 to 2.8]; p < 0.001), and absence of capsular attachments to the head fragment (OR 2.2 [95% CI 1.6 to 2.9]; p < 0.001) were independently associated with poor shoulder function and the occurrence of complications such as nonunion and avascular necrosis. CONCLUSION: Internal fixation of three-part and four-part proximal humerus fracture dislocations resulted in poor shoulder function and complications in a high number of patients, although fracture union was achieved in most patients. A nonunion proportion of 8%, 21% proportion of avascular necrosis, and 15% proportion of patients who underwent revision surgery suggests this is a fairly terrible injury. Being a woman and injury factors such as four-part fracture dislocation, absent metaphyseal head extension and back-bleeding from the head, height of the fractured head segment < 2 cm, and absence of capsular attachments to the head were independently associated with poor function and complications. Our findings can help surgeons decide between internal fixation and arthroplasty for the surgical treatment of these injuries in patients across different age groups and functional demands. LEVEL OF EVIDENCE: Level III, therapeutic study.

Probing Biothiols Using a Red-Emitting Pyridoxal Derivative by Adopting Copper(II) Displacement Approach and Cell Imaging.

An aggregation-induced emission (AIE) active Schiff base L was obtained by reacting pyridoxal and 2-hydroxy-1-naphthaldehyde with p-phenylenediamine in two simple steps. The colorimetric, UV/VIS and fluorescence studies of L revealed that the yellow emissive L (λem =540 nm, λex =450 nm) in pure DMSO turned to a red-emissive L, when the poor solvent fraction (HEPES buffer, 10 mM, pH 7.4) was increased above 50 % in DMSO. The SEM and DLS results indicated the formation of self-aggregates of L that restricted the intramolecular motion and promoted the excited state intramolecular proton transfer (ESIPT) process. The cations sensing ability of the AIEgen L was explored in HEPES buffer (5 % DMSO, 10 mM, pH 7.4), where Cu2+ selectively quenched the fluorescence at 608 nm due to the chelation-enhanced fluorescence quenching (CHEQ) effect with an estimated sensitivity limit of 0.9 µM. Subsequently, the in situ formed AIEgen L-Cu2+ complex was applied for the cascade detection of glutathione (GSH), cysteine (Cys) and homocysteine (Hcy). The decomplexation of Cu2+ from the AIEgen L-Cu2+ by GSH, Cys and Hcy restored the quenched fluorescence emission of AIEgen L at 608 nm. With this Cu2+ displacement approach, the concentration of Cys, Hcy and GSH can be detected down to 2.8 µM, 3.12 µM and 2.0 µM, respectively. The practical utility of AIEgen L and AIEgen L-Cu2+ was examined by monitoring the selective analytes in real environmental and biological samples, and also applied successfully for the cell imaging applications.

The utility of 2-[18F]FDG-PET/CT maximum SUV versus CT attenuation for directing bone biopsies.

OBJECTIVES: This study aimed to compare the accuracy of PET/CT parameters with CT parameters for directing bone biopsies. METHODS: The study was an IRB-approved retrospective study of 388 patients who underwent both 2-[18F] FDG PET/CT and CT within 6 weeks before a bone biopsy. Age, sex, cancer type, lesion length, SUVmax, tumor to liver (T/L) ratio, CT attenuation, difference in CT attenuation between the lesion and normal bone (delta CT attenuation), and the absolute delta CT attenuation were used as predictors. T tests and chi-squared tests were used to compare variables. DeLong's test was used to compare receiver operator characteristic (ROC) curves. RESULTS: We reviewed the data from 388 patients. Of these, 295 patients had bone lesion biopsies, and 93 patients had bone marrow aspirations/biopsies. Biopsies of larger bone lesions (p = 0.033) and bone lesions with higher SUVmax (p = 0.005) were more likely to show malignancy. For bone lesions, the ROC curve for the SUVmax (AUC = 0.6827) was better than the ROC curves for delta CT attenuation (AUC = 0.5766, p = 0.032) and absolute delta CT attenuation (AUC = 0.5491, p = 0.006), but not significantly better than the ROC curves for CT attenuation (AUC = 0.5894, p = 0.061) and T/L ratio (AUC = 0.6778, p = 0.774). A threshold SUVmax of 5.25 had an accuracy of 0.713, sensitivity of 0.766, and specificity of 0.549 to predict malignancy in bone lesion biopsies. None of these variables predicted malignancy in bone marrow biopsies (p > 0.05 for all). CONCLUSIONS: Metabolic 2-[18F]FDG PET/CT parameters have more clinical impact for planning bone biopsies as compared to CT parameters. KEY POINTS: • The 2-[18F]FDG PET/CT measurement (SUVmax) has more clinical impact for planning bone biopsies as compared to CT measurements. • Neither the change in CT attenuation of the lesion relative to normal bone nor the absolute value of this change was a significant predictor of malignancy. • 2-[18F]FDG PET/CT may have clinical benefit and an additional role in directing bone biopsies.

What Is the Likelihood of Union After Coronal Limb Realignment Using Revision Osteosynthesis and Concurrent TKA in Patients with Advanced Arthritis and Loss of Fixation After Distal Metaphyseal Femur Fractures?

BACKGROUND: Metaphyseal fracture healing in the distal femur requires a stable biomechanical environment. The presence of arthritis-induced coronal-plane knee deformities can cause deviation of the mechanical axis, which results in asymmetric loading and increased bending forces in fractures of the distal femur metaphysis. This predisposes patients to nonunions or loss of fixation. Concurrent TKA during revision osteosynthesis might facilitate fracture healing, owing to its ability to correct coronal alignment, thereby restoring normal loading patterns at the fracture site, but to our knowledge, this has not been studied. QUESTIONS/PURPOSES: (1) Does TKA with concurrent revision internal fixation achieve fracture union in patients with coronal-plane deformity from knee arthritis and nonunion or loss of fixation in distal metaphyseal femoral fractures? (2) What is the survivorship and what are the short-term functional outcomes after these reconstructions? (3) What complications occur after these reconstructions? METHODS: Between 2015 and 2018, one surgeon treated 16 patients with a distal metaphyseal femur fracture nonunion and/or loss of fixation using concurrent TKA plus revision internal fixation. Autologous iliac crest bone grafting was performed in five patients with evident gaps at the fracture site. The indications for the procedure included patients older than 55 years of age presenting with a nonunion and/or loss of fixation of a distal metaphyseal femur fracture in the setting of painful Ahlbäck Grade III to V knee arthritis with an associated coronal-plane deformity. All patients meeting these indications were treated with this approach during the study period. Fracture union assessed by radiological bridging of at least three cortices, TKA survival free from revision due to any reason, coronal-plane correction using tibiofemoral angle, and patient mobility status assessed presurgery and at follow-up using the Parker mobility score (scored 0-9 points, with 9 indicating best mobility) were assessed by two surgeons who were not involved in the care of the study patients. Immediate and delayed complications were recorded. Patients were followed for a minimum of 24 months. The median (range) follow-up was 38 months (27 to 52 months). RESULTS: All fractures united after concurrent TKA plus revision internal fixation. In all, 14 of 16 fractures healed before 5 months, while the remaining two fractures united by 6 months. Survivorship analysis revealed a TKA component survival of 94% (95% CI 63% to 99%) at 52 months. The median (range) preoperative Parker mobility score of 5 points (3 to 8) improved to 7 points (2 to 9) at 12 months postoperatively and was maintained at last follow-up (p = 0.001). Four patients experienced complications; these were (1) prolonged surgical wound drainage resulting in debridement and polyethylene liner exchange, (2) deep knee infection needing a staged revision, (3) popliteal vein thrombosis, and (4) prolonged graft site pain. CONCLUSION: Concurrent TKA plus revision internal fixation is effective for achieving union in patients with distal metaphyseal femur nonunion and loss of fixation in the setting of coronal-plane deformity induced by knee arthritis. Short-term TKA survival and improvement in patient mobility are excellent, although 4 of 16 patients in this report experienced complications, as one might expect with a procedure of this magnitude. Based on our results, correction of arthritis-induced coronal-plane knee malalignment can be considered part of the surgical strategy when treating such distal metaphyseal femur nonunions. Better preoperative evaluation of the deformity and control-based comparative studies can further validate the utility of this technique. LEVEL OF EVIDENCE: Level II, therapeutic study.

Unscheduled DNA synthesis leads to elevated uracil residues at highly transcribed genomic loci in Saccharomyces cerevisiae.

Recombination and mutagenesis are elevated by active transcription. The correlation between transcription and genome instability is largely explained by the topological and structural changes in DNA and the associated physical obstacles generated by the transcription machinery. However, such explanation does not directly account for the unique types of mutations originating from the non-canonical residues, uracil or ribonucleotide, which are also elevated at highly transcribed regions. Based on the previous findings that abasic (AP) lesions derived from the uracil residues incorporated into DNA in place of thymine constitute a major component of the transcription-associated mutations in yeast, we formed the hypothesis that DNA synthesis ensuing from the repair of the transcription-induced DNA damage provide the opportunity for uracil-incorporation. In support of this hypothesis, we show here the positive correlation between the level of transcription and the density of uracil residues in the yeast genome indirectly through the mutations generated by the glycosylase that excise undamaged cytosine as well as uracil. The higher uracil-density at actively transcribed regions is confirmed by the long-amplicon PCR analysis. We also show that the uracil-associated mutations at a highly transcribed region are elevated by the induced DNA damage and reduced by the overexpression of a dUTP-catalyzing enzyme Dut1 in G1- or G2-phases of the cell cycle. Overall, our results show that the DNA composition can be modified to include higher uracil-content through the non-replicative, repair-associated DNA synthesis.

Enhancing respiratory sinus arrhythmia increases cardiac output in rats with left ventricular dysfunction.

KEY POINTS: Respiratory sinus arrhythmia is physiological pacing of the heart that disappears in cardiovascular disease and is associated with poor cardiac prognosis. In heart failure, cardiac pacing has little, if any, variation in rate at rest. We proposed that reinstatement of respiratory sinus arrhythmia would improve cardiac function in rats with heart failure. Heart failure rats were paced daily for 2 weeks with either respiratory sinus arrhythmia or paced monotonically at a matched heart rate; cardiac function was measured using non-invasive echocardiography. Cardiac output and stroke volume were increased in rats paced with respiratory sinus arrhythmia compared to monotonic pacing, via improvement in systolic function that persisted beyond the pacing treatment period. We propose that respiratory sinus arrhythmia pacing reverse-remodels the heart in heart failure and is worth considering as a new form of cardiac pacemaking. ABSTRACT: Natural pacing of the heart results in heart rate variability, an indicator of good health and cardiac function. A contributor to heart rate variability is respiratory sinus arrhythmia or RSA - an intrinsic respiratory modulated pacing of heart rate. The loss of RSA is associated with poor cardiac prognosis and sudden cardiac death. We tested if reinstatement of respiratory-modulated heart rate (RMH) would improve cardiac performance in heart failure. Heart failure was induced in Wistar rats by ligation of the left anterior descending coronary artery. Rats were unpaced, monotonically paced and RMH paced; the latter had the same average heart rate as the monotonically paced animals. Cardiac function was assessed non-invasively using echocardiography before and after 2 weeks of daily pacing at a time when pacing was turned off. RMH increased cardiac output by 20 ± 8% compared to monotonic pacing (-3 ± 5%; P < 0.05). This improvement in cardiac output was associated with an increase in stroke volume compared to monotonic pacing (P = 0.03) and improvement in circumferential strain (P = 0.02). Improvements in ejection fraction (P = 0.08) and surrogate measures of left ventricle compliance did not reach significance. Increases in contractility (P < 0.05) and coronary blood flow (P < 0.05) were seen in vitro during variable pacing to mimic RMH. Thus, in rats with left ventricular dysfunction, chronic RMH pacing improved cardiac function through improvements in systolic function. As these improvements were made with pacing switched off, we propose the novel idea that RMH pacing causes reverse-remodelling.

Genome-wide mapping of regions preferentially targeted by the human DNA-cytosine deaminase APOBEC3A using uracil-DNA pulldown and sequencing.

Activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic subunit (APOBEC) enzymes convert cytosines to uracils, creating signature mutations that have been used to predict sites targeted by these enzymes. Mutation-based targeting maps are distorted by the error-prone or error-free repair of these uracils and by selection pressures. To directly map uracils created by AID/APOBEC enzymes, here we used uracil-DNA glycosylase and an alkoxyamine to covalently tag and sequence uracil-containing DNA fragments (UPD-Seq). We applied this technique to the genome of repair-defective, APOBEC3A-expressing bacterial cells and created a uracilation genome map, i.e. uracilome. The peak uracilated regions were in the 5'-ends of genes and operons mainly containing tRNA genes and a few protein-coding genes. We validated these findings through deep sequencing of pulldown regions and whole-genome sequencing of independent clones. The peaks were not correlated with high transcription rates or stable RNA:DNA hybrid formation. We defined the uracilation index (UI) as the frequency of occurrence of TT in UPD-Seq reads at different original TC dinucleotides. Genome-wide UI calculation confirmed that APOBEC3A modifies cytosines in the lagging-strand template during replication and in short hairpin loops. APOBEC3A's preference for tRNA genes was observed previously in yeast, and an analysis of human tumor sequences revealed that in tumors with a high percentage of APOBEC3 signature mutations, the frequency of tRNA gene mutations was much higher than in the rest of the genome. These results identify multiple causes underlying selection of cytosines by APOBEC3A for deamination, and demonstrate the utility of UPD-Seq.

Simultaneous Monitoring of Crystalline Active Materials and Resistance Evolution in Lithium-Sulfur Batteries.

Operando X-ray diffraction (XRD) is a valuable tool for studying secondary battery materials as it allows for the direct correlation of electrochemical behavior with structural changes of crystalline active materials. This is especially true for the lithium-sulfur chemistry, in which energy storage capability depends on the complex growth and dissolution kinetics of lithium sulfide (Li2S) and sulfur (S8) during discharge and charge, respectively. In this work, we present a novel development of this method through combining operando XRD with simultaneous and continuous resistance measurement using an intermittent current interruption (ICI) method. We show that a coefficient of diffusion resistance, which reflects the transport properties in the sulfur/carbon composite electrode, can be determined from analysis of each current interruption. Its relationship to the established Warburg impedance model is validated theoretically and experimentally. We also demonstrate for an optimized electrode formulation and cell construction that the diffusion resistance increases sharply at the discharge end point, which is consistent with the blocking of pores in the carbon host matrix. The combination of XRD with ICI allows for a direct correlation of structural changes with not only electrochemical properties but also energy loss processes at a nonequilibrium state and, therefore, is a valuable technique for the study of a wide range of energy storage chemistries.

Indian orthopaedics: the past, present, and future.

The editorial summarizes the Indian orthopaedic history in brief and provides an overview of the articles to be published in the Indian traumatology edition.

Valgus intertrochanteric osteotomy for femur neck pseudoarthrosis: a simple solution to a complex problem that has stood the test of time.

Femoral neck nonunion in young patients has always been a difficult problem to deal with for surgeons. Numerous surgical procedures to address either the biological or mechanical issues at the nonunion have been described and most of them have been associated with variable results. Isolated biological augmentation is often associated with poor results and some techniques like vascularized grafting may require not so commonly available expertise. Valgus osteotomy is aimed to correct the abnormal fracture biomechanics associated with femoral neck fractures. By altering the nature of force transmission across the nonunion, shear forces are converted into compressive forces that lead to rapid osseous union without the need for bone grafting. Though the principles are sound and were described a long time ago, the technical aspects have evolved over time. Various modifications have been described to overcome shortcomings such as limb length discrepancy, reduction of femoral offset, alteration in mechanical axis, and the overall proximal femur anatomy. In this review, we look back at the fundamental principles and recent literature on the results of valgus intertrochanteric osteotomy for femoral neck pseudoarthrosis. We also highlight the important need for accurate preoperative planning and surgical execution. Lastly, we elaborate on the technical improvisations that have happened over time in order to improve functional results and to minimize complications and poor outcome after a valgus osteotomy.