Two-year follow-up of transcatheter aortic valve replacement in low-risk patients with symptomatic severe bicuspid aortic valve stenosis.

BACKGROUND: In 2019, the US Food and Drug Administration (FDA) approved transcatheter aortic valve replacement (TAVR) for low-risk patients with symptomatic severe tricuspid aortic stenosis. However, bicuspid aortic valve (BAV) patients were included only in single-arm registries of pivotal low-risk TAVR trials, resulting in limited data for this subgroup. METHODS: The LRT (Low Risk TAVR) trial was an investigator-initiated, prospective, multicenter study and the first FDA-approved investigational device exemption trial to evaluate the feasibility of TAVR with balloon-expandable or self-expanding valves in low-risk patients with symptomatic severe BAV stenosis. This analysis reports 2-year follow-up, assessing the primary outcome of all-cause mortality and evaluating clinical outcomes. RESULTS: From 2016 to 2019, a total of 72 low-risk patients diagnosed with symptomatic, severe BAV stenosis underwent TAVR across six centers. Six patients were lost to follow-up. At 2-year follow-up, mortality was 1.5% (1 of 66 patients). Among the remaining 65 patients, four experienced nondisabling strokes (6.2%), while 2 (3.1%) developed infective endocarditis. No new permanent pacemakers were required beyond the 30-day follow-up, and no patients, including those with endocarditis, needed aortic valve re-intervention. At the 2-year echocardiography follow-up (n = 65), 27.8% of BAV patients showed mild aortic regurgitation, with none exhibiting moderate or severe regurgitation. The mean aortic gradient was 12.1 ± 4.1 mmHg, and the mean valve area was 1.7 ± 0.5 cm². CONCLUSION: The 2-year follow-up confirms commendable clinical outcomes of TAVR in patients with bicuspid aortic stenosis, establishing its evident safety.

The UT Health Living Room: Expanding the Psychiatric Crisis Continuum of Care.

Traditional forms of psychiatric crisis treatment increasingly are being buttressed by services along the Psychiatric Crisis Continuum of Care, such as short-term crisis stabilization services and peer crisis services. The UT Health Living Room (LR) is an outpatient crisis counseling service that adds three promising elements to the Continuum: (1) it integrates outpatient treatment plans into crisis counseling, (2) provides care in a space and with staff who are familiar to patients, and (3) provides training in evidence-based crisis intervention. We examined two-year LR feasibility and outcome data. Mixed-method analyses used longitudinal clinic data and patient self-report measures. Results provide initial support for the feasibility, cost effectiveness and clinical effectiveness of the LR. Limitations include non-blinded ratings, limited experimental control, and simple cost-effectiveness methodology. The UT Living Room is feasible and offers novel elements to help patients in community clinics address emotional crises.

Successful Extracorporeal Cardiopulmonary Resuscitation Despite Aortic Occlusion.

We present the case of a 62-year-old man with severe coronary artery disease who presented to the hospital in refractory ventricular fibrillation cardiac arrest. He showed signs of life despite prolonged resuscitation. We thus decided to initiate extracorporeal cardiopulmonary resuscitation (ECPR). The patient had a known total occlusion of his infrarenal aorta that had been surgically bypassed with a bifemoral-axillary graft. We successfully initiated ECPR via the surgical graft, establishing blood flow to the central circulation through the axillary artery in a peripheral configuration while ensuring blood flow to the left leg via the femoral-femoral graft. The patient was extubated neurologically intact the following day and subsequently underwent coronary artery bypass graft surgery while on extracorporeal membrane oxygenation (ECMO) support. He was subsequently weaned off inotropic support and decannulated from ECMO. He was discharged home neurologically intact and independent in his activities of daily living. This case demonstrates that cannulation for ECPR via a surgical vascular graft is possible and that a total occlusion of the infrarenal aorta in the presence of a surgical bypass is not an absolute contraindication to ECMO.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Top-down proteomics.

Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine. This Primer explores TDP, including the underlying principles, recent advances and an outlook on the future. The experimental section discusses instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques and data collection. The results section looks at how to decipher raw data, visualize intact protein spectra and unravel data analysis. Additionally, proteoform identification, characterization and quantification are summarized, alongside approaches for statistical analysis. Various applications are described, including the human proteoform project and biomedical, biopharmaceutical and clinical sciences. These are complemented by discussions on measurement reproducibility, limitations and a forward-looking perspective that outlines areas where the field can advance, including potential future applications.

Misexpression of inactive genes in whole blood is associated with nearby rare structural variants.

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression.

Outbreaks of H5N1 High Pathogenicity Avian Influenza in South Africa in 2023 Were Caused by Two Distinct Sub-Genotypes of Clade 2.3.4.4b Viruses.

In 2023, South Africa continued to experience sporadic cases of clade 2.3.4.4b H5N1 high-pathogenicity avian influenza (HPAI) in coastal seabirds and poultry. Active environmental surveillance determined that H5Nx, H7Nx, H9Nx, H11Nx, H6N2, and H12N2, amongst other unidentified subtypes, circulated in wild birds and ostriches in 2023, but that H5Nx was predominant. Genome sequencing and phylogenetic analysis of confirmed H5N1 HPAI cases determined that only two of the fifteen sub-genotypes that circulated in South Africa in 2021-2022 still persisted in 2023. Sub-genotype SA13 remained restricted to coastal seabirds, with accelerated mutations observed in the neuraminidase protein. SA15 caused the chicken outbreaks, but outbreaks in the Paardeberg and George areas, in the Western Cape province, and the Camperdown region of the KwaZulu-Natal province were unrelated to each other, implicating wild birds as the source. All SA15 viruses contained a truncation in the PB1-F2 gene, but in the Western Cape SA15 chicken viruses, PA-X was putatively expressed as a novel isoform with eight additional amino acids. South African clade 2.3.4.4b H5N1 viruses had comparatively fewer markers of virulence and pathogenicity compared to European strains, a possible reason why no spillover to mammals has occurred here yet.

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Genome-wide meta-analyses of restless legs syndrome yield insights into genetic architecture, disease biology and risk prediction.

Restless legs syndrome (RLS) affects up to 10% of older adults. Their healthcare is impeded by delayed diagnosis and insufficient treatment. To advance disease prediction and find new entry points for therapy, we performed meta-analyses of genome-wide association studies in 116,647 individuals with RLS (cases) and 1,546,466 controls of European ancestry. The pooled analysis increased the number of risk loci eightfold to 164, including three on chromosome X. Sex-specific meta-analyses revealed largely overlapping genetic predispositions of the sexes (rg = 0.96). Locus annotation prioritized druggable genes such as glutamate receptors 1 and 4, and Mendelian randomization indicated RLS as a causal risk factor for diabetes. Machine learning approaches combining genetic and nongenetic information performed best in risk prediction (area under the curve (AUC) = 0.82-0.91). In summary, we identified targets for drug development and repurposing, prioritized potential causal relationships between RLS and relevant comorbidities and risk factors for follow-up and provided evidence that nonlinear interactions are likely relevant to RLS risk prediction.

Increased scalability and sequencing quality of an epigenetic age prediction assay.

Epigenetic ageing in a human context, has been used to better understand the relationship between age and factors such as lifestyle and genetics. In an ecological setting, it has been used to predict the age of individual animals for wildlife management. Despite the importance of epigenetic ageing in a range of research fields, the assays to measure epigenetic ageing are either expensive on a large scale or complex. In this study, we aimed to improve the efficiency and sequencing quality of an existing epigenetic ageing assay for the Australian Lungfish (Neoceratodus forsteri). We used an enzyme-based alternative to bisulfite conversion to reduce DNA fragmentation and evaluated its performance relative to bisulfite conversion. We found the sequencing quality to be 12% higher with the enzymatic alternative compared to bisulfite treatment (p-value < 0.01). This new enzymatic based approach, although currently double the cost of bisulfite treatment can increases the throughput and sequencing quality. We envisage this assay setup being adopted increasingly as the scope and scale of epigenetic ageing research continues to grow.

Identifying potential provenances for climate-change adaptation using spatially variable coefficient models.

BACKGROUND: Selection of climate-change adapted ecotypes of commercially valuable species to date relies on DNA-assisted screening followed by growth trials. For trees, such trials can take decades, hence any approach that supports focussing on a likely set of candidates may save time and money. We use a non-stationary statistical analysis with spatially varying coefficients to identify ecotypes that indicate first regions of similarly adapted varieties of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) in North America. For over 70,000 plot-level presence-absences, spatial differences in the survival response to climatic conditions are identified. RESULTS: The spatially-variable coefficient model fits the data substantially better than a stationary, i.e. constant-effect analysis (as measured by AIC to account for differences in model complexity). Also, clustering the model terms identifies several potential ecotypes that could not be derived from clustering climatic conditions itself. Comparing these six identified ecotypes to known genetically diverging regions shows some congruence, as well as some mismatches. However, comparing ecotypes among each other, we find clear differences in their climate niches. CONCLUSION: While our approach is data-demanding and computationally expensive, with the increasing availability of data on species distributions this may be a useful first screening step during the search for climate-change adapted varieties. With our unsupervised learning approach being explorative, finely resolved genotypic data would be helpful to improve its quantitative validation.

Ultrasound- and Doppler-Guided WALANT Arthroscopic Surgery for Patellar Tendinopathy with Partial Rupture in Elite Athletes-A 2-Year Follow-Up of a Prospective Case Series.

Background and Objectives: Patellar tendinopathy is difficult to treat, and when combined with partial rupture, there are additional challenges. The aim of this study was to evaluate the subjective outcome and return-to-sport status after ultrasound (US)- and colour doppler (CD)-guided wide awake local anaesthetic no tourniquet (WALANT) arthroscopic shaving in elite athletes. Material and Methods: Thirty Swedish and international elite athletes (27 males) with a long duration (>1 year) of persistent painful patellar tendinopathy in 35 patellar tendons, not responding to non-surgical treatment, were included. All patients were treated with the same protocol of arthroscopic shaving, including bone removal and debridement of partial rupture, followed by at least 3 months of structured rehabilitation. The VISA-P score and a study-specific questionnaire evaluating physical activity level and subjective satisfaction with the treatment were used for evaluation. Results: At the 2-year follow-up (mean 23, range 8-38 months), 25/30 patients (29/35 tendons) were satisfied with the treatment result and had returned to their pre-injury sport. The mean VISA-P score increased from 37 (range 7-69) before surgery to 80 (range 44-100) after surgery (p < 0.05). There was one drop-out (one tendon). There were no complications. Conclusions: US- and CD-guided WALANT arthroscopic shaving for persistent painful patellar tendinopathy, including bone removal and debridement of partial rupture, followed by structured rehabilitation showed good clinical results in the majority of the elite-level athletes.

Intravenous iron for acute and chronic heart failure with reduced ejection fraction (HFrEF) patients with iron deficiency: An updated systematic review and meta-analysis.

Patients with heart failure (HF) and iron deficiency are at increased risk of adverse clinical outcomes. We searched databases for randomised controlled trials that compared IV iron to placebo, in patients with HF with reduced ejection fraction (HFrEF). A total of 7,813 participants, all having HFrEF with 3,998 receiving IV iron therapy, and 3,815 control recipients were included. There was a significant improvement in Kansas City Cardiomyopathy Questionnaire favouring IV iron with MD 7.39, 95% CI [3.55, 11.22], p = 0.0002. Subgroup analysis, based on acute and chronic HF, has displayed a sustained statistical significance. Additionally, a significant increase in the left ventricular ejection fraction % was observed, with MD 3.76, 95% CI [2.32, 5.21], p < 0.00001. A significant improvement in 6-min walk test was noted, with MD 34.87, 95% CI [20.02, 49.72], p < 0.00001. Furthermore, IV iron showed significant improvement in NYHA class, peak VO2, serum ferritin, and haemoglobin levels. Finally, despite the lack of difference in terms of all-cause hospitalisation and HF-related death, IV iron was associated with a significant reduction in HF-related, any cardiovascular reason hospitalisations, and all-cause death; which supports the need for implementation of IV iron as a standard of care in patients with HF and iron deficiency.

Tendinopathic Plantaris but Normal Achilles Tendon Found in About One-Fifth of Patients Not Responding to Conservative Achilles Tendon Management - Results from a Prospective WALANT Surgical Case Series on 105 Tendons.

Purpose: Midportion Achilles tendinopathy is a relatively common condition. This study aimed to investigate the presence of a normal Achilles tendon, but a tendinopathic plantaris tendon, in a large and consecutive prospective sample of patients referred to a specialised tendon clinic for midportion Achilles tendon pain not responding to non-surgical treatment. Patients and Methods: A total of 105 consecutive tendons were operated on in 81 patients (62 males) suffering from painful midportion Achilles tendon pain. Clinical examination, ultrasound (US) and colour Doppler (CD) examination, and wide awake local anaesthetic no tourniquet (WALANT) surgery were performed in all patients. Results: For 19/105 (18%) tendons from 14 patients, clinical examination suspected plantaris tendinopathy alone as there was a distinct tenderness on the medial side, but no thickening of the Achilles tendon. US examination followed by surgery confirmed the diagnosis. Conclusion: Midportion Achilles tendon pain is not always related to Achilles tendinopathy since pain related to the plantaris tendon alone was found in almost every fifth patient. Consequently, there is an obvious need for proper examination to identify the pain source and establish a correct diagnosis before treatment.

Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis.

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis. In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

Real-World Outcomes for the Fifth-Generation Balloon Expandable Transcatheter Heart Valve in the United States.

BACKGROUND: The fifth-generation SAPIEN 3 Ultra Resilia valve (S3UR) incorporates several design changes as compared with its predecessors, the SAPIEN 3 (S3) and SAPIEN 3 Ultra (S3U) valves, including bovine leaflets treated with a novel process intended to reduce structural valve deterioration via calcification, as well as a taller external skirt on the 29-mm valve size to reduce paravalvular leak (PVL). The clinical performance of S3UR compared with S3 and S3U in a large patient population has not been previously reported. OBJECTIVES: The aim of this study was to compare S3UR to S3/S3U for procedural, in-hospital, and 30-day clinical and echocardiographic outcomes after transcatheter aortic valve replacement (TAVR). METHODS: Patients enrolled in the STS/ACC TVT (Society of Thoracic Surgeons/American College of Cardiology Transcatheter Valve Therapy) Registry between January 1, 2021, and June 30, 2023, who underwent TAVR with S3UR or S3U/S3 valve platforms were propensity-matched and evaluated for procedural, in-hospital, and 30-day clinical and echocardiographic outcomes. RESULTS: 10,314 S3UR patients were propensity matched with 10,314 patients among 150,539 S3U/S3 patients. At 30 days, there were no statistically significant differences in death, stroke, or bleeding, but a numerically higher hospital readmission rate in the S3UR cohort (8.5% vs 7.7%; P = 0.04). At discharge, S3UR patients exhibited significantly lower mean gradients (9.2 ± 4.6 mm Hg vs 12.0 ± 5.7 mm Hg; P < 0.0001) and larger aortic valve area (2.1 ± 0.7 cm2 vs 1.9 ± 0.6 cm2; P < 0.0001) than patients treated with S3/S3U. The 29-mm valve size exhibited significant reduction in mild PVL (5.3% vs 9.4%; P < 0.0001). CONCLUSIONS: S3UR TAVR is associated with lower mean gradients and lower rates of PVL than earlier generations of balloon expandable transcatheter heart valve platforms.

Directed evolution of genetically encoded LYTACs for cell-mediated delivery.

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.

Effects of a humanized CD47 antibody and recombinant SIRPα proteins on triple negative breast carcinoma stem cells.

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca2+) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.