A B-Cell-Specific Enhancer Orchestrates Nuclear Architecture to Generate a Diverse Antigen Receptor Repertoire.

The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.

Cutting Edge: Proper Orientation of CTCF Sites in Cer Is Required for Normal Jκ-Distal and Jκ-Proximal Vκ Gene Usage.

Igκ locus contraction and Vκ gene usage are controlled by Cer, a cis-acting sequence in the Vκ-Jκ intervening region. This effect is attributed to two CTCF-binding sites within Cer that are oriented toward the Vκ gene region. However, the importance of Cer CTCF orientation in regulating VκJκ rearrangement is unknown. We used CRISPR/Cas9 editing to delete and invert Cer in murine Abl pro-B cell lines. This revealed that Cer orientation is critical because clones with either an inverted or deleted Cer element show skewing toward Jκ-proximal Vκ gene usage. However, only Cer deletion increased Jκ-proximal Vκ germline transcription, suggesting an insulating function of Cer. Lastly, circularized chromosome conformation capture interaction data show that Cer CTCF orientation regulates long-range interactions with inversion clones displaying fewer interactions with regions in the middle and distal parts of the Vκ locus and more interactions to downstream regions compared with wild-type or deletion clones.

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution.

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 µs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of ß-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.

Prophylactic emicizumab for hemophilia A in the Asia-Pacific region: A randomized study (HAVEN 5).

Background: Emicizumab is a subcutaneously administered humanized, bispecific, monoclonal antibody approved for prophylaxis in people with hemophilia A. Methods: HAVEN 5 (NCT03315455) is a randomized, open-label, phase 3 study of individuals aged ≥12 years with severe hemophilia A without factor VIII (FVIII) inhibitors, or hemophilia A of any severity with FVIII inhibitors, across the Asia-Pacific region. Participants were randomly assigned (2:2:1) to receive emicizumab 1.5 mg/kg once weekly (arm A), emicizumab 6 mg/kg every 4 weeks (arm B), or no prophylaxis (arm C). The primary end point was annualized bleeding rate (ABR) for treated bleeds; ABRs were compared between people receiving emicizumab prophylaxis versus those with no prophylaxis. Secondary end points included ABR for treated target joint bleeds. Safety was also evaluated. Results: From April 26, 2018, to January 4, 2019, 70 of 76 screened participants were enrolled and randomized (arm A, n = 29; arm B, n = 27; arm C, n = 14). ABRs (95% confidence interval) for treated bleeds and treated target joint bleeds, respectively, were: arm A, 1.0 (0.53-1.85) and 0.4 (0.18-1.09); arm B, 1.0 (0.50-1.84) and 0.3 (0.12-0.85); arm C, 27.0 (13.29-54.91) and 8.6 (3.15-23.42). The most common adverse event, upper respiratory tract infection, was reported for 14 of 56 (25.0%; emicizumab) and 2 of 14 (14.3%; no prophylaxis) participants. No thrombotic events, thrombotic microangiopathies, or deaths were reported. Conclusion: Emicizumab 1.5 mg/kg once weekly and 6 mg/kg every 4 weeks demonstrated bleed control in this study population, was well tolerated, and could improve use of prophylaxis in people with hemophilia A.

Baseline global longitudinal strain by computed tomography is associated with post transcatheter aortic valve replacement outcomes.

BACKGROUNDS: Subclinical myocardial dysfunction detected by global longitudinal strain (GLS) using echocardiography is associated with poor outcomes in patients with severe aortic stenosis (AS) despite normal left ventricular ejection fraction (LVEF). Computed tomography angiography derived GLS (CTA-GLS) has recently shown to be feasible, however the prognostic value remains unclear in severe AS patients treated with transcatheter aortic valve replacement (TAVR). METHODS: We analyzed consecutive patients who underwent TAVR with pre-TAVR retrospective gated acquisition CTA study with adequate image quality covering the entire left ventricle. CTA-GLS analysis was performed using 2D CT-Cardiac Performance Analysis prototype software (TomTec GmbH). Kaplan-Meier and Cox regression analyses were performed to evaluate the association of baseline CTA-GLS with all-cause mortality and a composite outcome of all-cause death and hospitalization for heart failure after TAVR. RESULTS: A total of 223 patients were included (mean age 83.5 ± 6.8 years, 45.7% female, mean CTA-LVEF 50.7 ± 14.5%). During a median follow-up of 32 months, 81 all-cause deaths and 134 composite outcomes occurred. When compared to patients with normal LVEF (≥50%) and preserved CTA-GLS (≤-20.5%), patients with normal LVEF but reduced CTA-GLS (>-20.5%) had higher all-cause mortality (Chi-square 6.89, p = 0.032) and the risk of composite outcome (Chi-square 7.80, p = 0.020) which was no different than those with impaired LVEF. Reduced CTA-GLS was independently associated with all-cause mortality (HR 1.71, 95% CI 1.01-2.90, p = 0.049) and the risk of composite outcome (HR 1.51, 95% CI 1.01-2.25, p = 0.044) on multivariable Cox regression analysis. CONCLUSIONS: Reduced CTA-GLS provides independent prognostic value above multiple clinical and echocardiographic characteristics.

Global longitudinal strain assessment by computed tomography in severe aortic stenosis patients - Feasibility using feature tracking analysis.

BACKGROUND: Global longitudinal strain (GLS) detects subclinical myocardial changes in patients with aortic stenosis (AS). Although GLS is typically measured by transthoracic echocardiography (TTE), assessment by multiphasic gated computed tomography angiography (CTA) has become recently available. We sought to evaluate the feasibility of CTA-derived GLS assessment and compare its agreement with TTE using the same post-processing software in severe AS patients undergoing transcatheter aortic valve replacement (TAVR) evaluation. METHODS: We evaluated patients with severe AS, sinus rhythm and adequate image quality for GLS analysis by both CTA and TTE pre-TAVR using 2D CT-Cardiac Performance Analysis prototype software (TomTec). The 18-segment model was used for GLS analysis by averaging the three long-axis views in both CTA and TTE studies. Agreement was assessed using linear regression and Bland-Altman analysis. RESULTS: A total of 123 consecutive patients were included (mean age 84 ±â€¯7 years, 45% female). The mean left ventricular ejection fraction (LVEF) by CTA and TTE were similar 53 ±â€¯14% for both. On average, CTA-derived GLS was greater than by TTE (-20 ±â€¯6.5% vs. -16 ±â€¯4.9%, respectively, p < 0.001). There was a moderate correlation between GLS assessed by CTA vs. TTE (r = 0.62, p < 0.001), although variability between imaging methods existed. The correlation between GLS and LVEF was strong (r = -0.90, p < 0.001 for CTA, r = -0.88, p < 0.001 for TTE) using the same imaging modality. CONCLUSION: CTA-derived GLS assessment is feasible in selected patients with sinus rhythm and adequate image quality. The agreement of GLS between TTE and CTA is moderate but not interchangeable suggesting a potential modality-specific GLS threshold.

Outcomes of persistent pulmonary hypertension following transcatheter aortic valve replacement.

OBJECTIVES: To determine the prevalence and factors associated with persistent pulmonary hypertension (PH) following transcatheter aortic valve replacement (TAVR) and its relationship with long-term mortality. METHODS: Consecutive patients who underwent TAVR from July 2011 through January 2016 were studied. The prevalence of baseline PH (mean pulmonary artery pressure ≥25 mm Hg on right heart catheterisation) and the prevalence and the predictors of persistent≥moderate PH (pulmonary artery systolic pressure (PASP)>45 mm Hg on 1 month post-TAVR transthoracic Doppler echocardiography) were collected. Cox models quantified the effect of persistent PH on subsequent mortality while adjusting for confounders. RESULTS: Of the 407 TAVR patients, 273 (67%) had PH at baseline. Of these, 102 (25%) had persistent≥moderate PH. Mortality at 2 years in patients with no baseline PH versus those with PH improvement (follow-up PASP≤45 mm Hg) versus those with persistent≥moderate PH was 15.4%, 16.6% and 31.3%, respectively (p=0.049). After adjusting for Society of Thoracic Surgeons Predicted Risk of Mortality and baseline right ventricular function (using tricuspid annular plane systolic excursion), persistent≥moderate PH remained associated with all-cause mortality (HR=1.82, 95% CI 1.06 to 3.12, p=0.03). Baseline characteristics associated with increased likelihood of persistent≥moderate PH were ≥moderate tricuspid regurgitation, ≥moderate mitral regurgitation, atrial fibrillation/flutter, early (E) to late (A) ventricular filling velocities (E/A ratio) and left atrial volume index. CONCLUSIONS: Persistency of even moderate or greater PH at 1 month post-TAVR is common and associated with higher all-cause mortality.

New Zeno-Like Liquid States.

A Zeno line is a straight line in a fluid's temperature-density plane where the compressibility factor equals unity and stretches from supercritical to subcritical regimes. A second unexpected linearity also occurs in liquids in their normal liquid range (NLR) where the compressibility factor approaches zero. This Zeno-like line includes liquids as diverse as monatomic and diatomic elements to hydrogen bonding liquids, with water and helium being the only currently known exceptions. Remarkably, this second linearity is also present in polymeric liquids, molten metals, and salts, i.e., it is a generic characteristic of liquids with negligible or low vapor pressures. This observation yields the following new corresponding states principle: Saturated liquid densities are a linear function of temperature in the NLR for molecular, polymeric, inorganic, ionic, and metallic liquids and superpose to form a single master curve. Another Zeno-like linearity in the NLR has also been identified for the configurational energy of many liquids. Extension of this line to zero temperature defines the ground state configurational energy of the hypothetical disordered liquid (glass).