Assessment of antibody library diversity through next generation sequencing and technical error compensation

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.Funded by European Union Seventh Framework Program [grant No. 604102 A.C.] (Human Brain Project). https://www.humanbrainproject.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Outcomes of Left Bundle Branch Area Pacing for Cardiac Resynchronization Therapy: An Updated Systematic Review and Meta-analysis.

BACKGROUND: Real-world data on the use of left bundle branch area pacing (LBBAP) as an alternative novel pacing strategy to biventricular pacing (BVP) for cardiac resynchronization therapy (CRT) remains scarce. We aimed to investigate the outcomes of LBBAP as an alternative to BVP as a method of CRT. METHODS: Electronic databases were searched for studies on the use of LBBAP as CRT and studies that compared LBBAP with BVP. The main outcomes examined were changes in New York Heart Association classification, left ventricular end-diastolic diameter, left ventricular ejection fraction, and paced QRS duration post CRT device implantation. RESULTS: Our meta-analysis included 8 nonrandomized studies with a total of 527 patients who underwent LBBAP as CRT. In studies with a BVP comparison group, patients with LBBAP had a greater reduction in paced QRS (mean difference [MD], 27.91 msec; 95% confidence interval [CI], 22.33-33.50), and a greater improvement in New York Heart Association class (MD, 0.59; 95% CI, 0.28-0.90) and left ventricular ejection fraction (MD, 6.77%; 95% CI, 3.84-9.71). Patients with underlying left bundle branch block appeared to benefit the most from LBBAP compared with patients without underlying left bundle branch block. CONCLUSIONS: LBBAP might be a reasonable option for patients who meet indications for CRT, particularly in those who have limited anatomy or do not benefit from CRT. Randomized trials are needed to compare LBBAP with BVP for CRT and to identify which populations might benefit the most from LBBAP

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation

none16noCerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3′UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.openMartins M.; Galfre S.; Terrigno M.; Pandolfini L.; Appolloni I.; Dunville K.; Marranci A.; Rizzo M.; Mercatanti A.; Poliseno L.; Morandin F.; Pietrosanto M.; Helmer-Citterich M.; Malatesta P.; Vignali R.; Cremisi F.Martins, M.; Galfre, S.; Terrigno, M.; Pandolfini, L.; Appolloni, I.; Dunville, K.; Marranci, A.; Rizzo, M.; Mercatanti, A.; Poliseno, L.; Morandin, F.; Pietrosanto, M.; Helmer-Citterich, M.; Malatesta, P.; Vignali, R.; Cremisi, F