A new generation of nanobody research tools using improved mass spectrometry-based discovery methods.

Single-domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, therapeutic, and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research-i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat immunoglobulin G. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

The Evolution of Carotid Surgery.

This review traces the evolution of carotid surgery from its ancient roots to modern practice. The first significant appreciation of the carotid arteries' role in cerebral ischemia was made by Johann Jakob Wepfer in his 1658 book on apoplexy, which described the separate blood supply to each cerebral hemisphere by the carotid arteries. The 16th-19th centuries saw initial attempts at carotid ligation, while the 20th century marked significant advancements. The first carotid endarterectomy (CEA) by DeBakey, Cooley, and Eastcott in the 1950s paved the way for a new technique in carotid surgery and many subsequent refinements in surgical techniques related to CEA. The latter half of the 20th century brought innovations such as patch angioplasty, microsurgical techniques, and intraoperative shunting, significantly improving outcomes. Pivotal clinical trials established CEA as the standard treatment for symptomatic carotid stenosis. The emergence of carotid artery stenting (CAS) in the 1980s introduced a less invasive alternative, sparking ongoing debates about optimal treatment strategies. Recent developments include transcarotid artery revascularization (TCAR) and transfemoral CAS, with studies comparing their efficacy to traditional CEA. Current research focuses on plaque morphology and novel diagnostic factors to further personalize treatment strategies for carotid artery stenosis.

Bioprosthetic versus mechanical valves for mitral valve replacement in patients < 70 years: an updated pairwise meta-analysis.

BACKGROUND: The ideal conduit for mitral valve replacement (MVR) remains elusive, particularly among younger patients due to increased life expectancy. We perform a pairwise meta-analysis comparing the use of bioprosthetic valves (BPV) and mechanical mitral valves (MMV) in patients < 70 years old undergoing MVR. METHODS: We comprehensively searched medical databases to identify studies comparing the use of BPV and MMV in patients < 70 years old undergoing MVR. Pairwise meta-analysis was performed using the Mantel-Haenszel method in R version 4.0.2. Outcomes were pooled using the random effect model as risk ratios (RR) with their 95% confidence intervals (95% CI). RESULTS: 16,879 patients from 15 studies were pooled. Compared to MMV, BPV was associated with significantly higher rates of 30-day mortality (RR 1.53, p = 0.0006) but no difference in 30-day stroke (RR 0.70, p = 0.43). At a weighted mean follow-up duration of 14.1 years, BPV was associated with higher rates of long-term mortality (RR 1.28, p = 0.0054). No difference was seen between the two groups for risk of long-term stroke (RR 0.92, p = 0.67), reoperation(RR 1.72, p = 0.12), or major-bleeding (RR 0.57, p = 0.10) at a weighted mean follow-up duration of 11.7, 11.3, and 11.9 years, respectively. CONCLUSION: The use of MMV in patients < 70 undergoing MVR is associated with lower rates of 30-day/long-term mortality compared to BPV. No significant differences were observed for risk of 30-day/long-term stroke, long-term reoperation, and long-term major bleeding. These findings support the use of MMV in younger patients, although prospective, randomized trials are still needed.

Transcatheter versus surgical valve replacement in patients with bicuspid aortic valves: an updated meta-analysis.

Patients with bicuspid aortic valves (BAV) are predisposed to the development of aortic stenosis. We performed a pairwise meta-analysis, comparing the efficacy of transcatheter aortic valve replacement (TAVR) versus surgical aortic valve replacement (SAVR) in patients with BAV. Medical databases were queried to pool comparative studies of interest. Single-arm studies, conference presentations, animal studies, and studies that involved patients with tricuspid aortic morphology were excluded. Outcomes were pooled as risk ratios (RRs) with their 95% confidence intervals (CI) using the random effects model in R. There were 60,858 patients with BAV (7,565 TAVR, 53,293 SAVR) included. Compared with SAVR, TAVR was associated with a significantly lower risk of 30-day major bleeding (RR 0.29, 95%CI 0.13 to 0.63, p=0.01) but a higher risk of new permanent pacemaker placement (RR 2.17, 95%CI 1.03 to 4.58, p=0.04). No significant differences were seen with other explored outcomes, including 30-day/mid-term mortality, stroke, acute kidney injury, major vascular complications, paravalvular leak, and conduction abnormalities. In conclusion, in patients with BAV, TAVR is associated with a lower risk of 30-day major bleeding but has an increased risk for permanent pacemaker implantation when compared with SAVR. Future large-scale randomised trials comparing both the short-and long-term outcomes of SAVR and TAVR in patients with BAV are needed to assess the efficacy of each modality in a controlled population across long follow-up durations.

US physician perspective on the use of biomarker and ctDNA testing in patients with melanoma.

New treatments have increased survival of patients with melanoma, and methods to monitor patients throughout the disease process are needed. Circulating tumor DNA (ctDNA) is a predictive and prognostic biomarker that may allow routine, real-time monitoring of disease status. We surveyed 44 US physicians to understand their preferences and practice patterns for biomarker and ctDNA testing in their patients with melanoma. Tumor biomarker testing was often ordered in stage IIIA-IV patients. Barriers to biomarker testing include insufficient tissue (60%) and lack of insurance coverage (54%). ctDNA testing was ordered by 16-18% of physicians for stages II-IV. Reasons for not using ctDNA testing included lack of prospective data (41%), ctDNA testing used for research only (18%), and others. Physicians (≥74%) believed that ctDNA assays could help with monitoring and treatment selection throughout the disease process. Physicians consider ctDNA testing potentially valuable for clinical decision-making but cited concerns that should be addressed.

Molecular targets of statins and their potential side effects: Not all the glitter is gold.

Statins are a class of drugs widely used worldwide to manage hypercholesterolemia and the prevention of secondary heart attacks. Currently, available statins vary in terms of their pharmacokinetic and pharmacodynamic profiles. Although the primary target of statins is the inhibition of HMG-CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthesis, statins exhibit many pleiotropic effects downstream of the mevalonate pathway. These pleiotropic effects include the ability to reduce myocardial fibrosis, pathologic cardiac disease states, hypertension, promote bone differentiation, anti-inflammatory, and antitumor effects through multiple mechanisms. Although these pleiotropic effects of statins may be a cause for enthusiasm, there are many adverse effects that, for the most part, are unappreciated and need to be highlighted. These adverse effects include myopathy, new-onset type 2 diabetes, renal and hepatic dysfunction. Although these adverse effects may be relatively uncommon, considering the number of people worldwide who use statins daily, the actual number of people affected becomes quite large. Also, co-administration of statins with several other medications, herbal agents, and foods, which interact through common enzymatic pathways, can have untoward clinical consequences. In this review, we address these concerns.