Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9.

CRISPR-Cas9 is the forefront technology for editing the genome. In this system, the Cas9 protein is programmed with guide RNAs to process DNA sequences that match the guide RNA forming an RNA:DNA hybrid structure. However, the binding of DNA sequences that do not fully match the guide RNA can limit the applicability of CRISPR-Cas9 for genome editing, resulting in the so-called off-target effects. Here, molecular dynamics is used to probe the effect of DNA base pair mismatches within the RNA:DNA hybrid in CRISPR-Cas9. Molecular simulations revealed that the presence of mismatched pairs in the DNA at distal sites with respect to the Protospacer Adjacent Motif (PAM) recognition sequence induces an extended opening of the RNA:DNA hybrid, leading to novel interactions established by the unwound nucleic acids and the protein counterpart. On the contrary, mismatched pairs upstream of the RNA:DNA hybrid are rapidly incorporated within the heteroduplex, with minor effect on the protein-nucleic acid interactions. As a result, mismatched pairs at PAM distal ends interfere with the activation of the catalytic HNH domain, while mismatches fully embedded in the RNA:DNA do not affect the HNH dynamics and enable its activation to cleave the DNA. These findings provide a mechanistic understanding to the intriguing experimental evidence that PAM distal mismatches hamper a proper function of HNH, explaining also why mismatches within the heteroduplex are much more tolerated. This constitutes a step forward in understanding off-target effects in CRISPR-Cas9, which encourages novel structure-based engineering efforts aimed at preventing the onset of off-target effects

Potentiality in Biology

We take the potentialities that are studied in the biological sciences (e.g., totipotency) to be an important subtype of biological dispositions. The goal of this paper is twofold: first, we want to provide a detailed understanding of what biological dispositions are. We claim that two features are essential for dispositions in biology: the importance of the manifestation process and the diversity of conditions that need to be satisfied for the disposition to be manifest. Second, we demonstrate that the concept of a disposition (or potentiality) is a very useful tool for the analysis of the explanatory practice in the biological sciences. On the one hand it allows an in-depth analysis of the nature and diversity of the conditions under which biological systems display specific behaviors. On the other hand the concept of a disposition may serve a unificatory role in the philosophy of the natural sciences since it captures not only the explanatory practice of biology, but of all natural sciences. Towards the end we will briefly come back to the notion of a potentiality in biology

3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

INTRODUCTION: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. METHODS: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. RESULTS: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. DISCUSSION: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies

Intercomparison of shallow water bathymetry, hydro-optics, and benthos mapping techniques in Australian and Caribbean coastal environments

Science, resource management, and defense need algorithms capable of using airborne or satellite imagery to accurately map bathymetry, water quality, and substrate composition in optically shallow waters. Although a variety of inversion algorithms are available, there has been limited assessment of performance and no work has been published comparing their accuracy and efficiency. This paper compares the absolute and relative accuracies and computational efficiencies of one empirical and five radiative-transfer-based published approaches applied to coastal sites at Lee Stocking Island in the Bahamas and Moreton Bay in eastern Australia. These sites have published airborne hyperspectral data and field data. The assessment showed that (1) radiative-transfer-based methods were more accurate than the empirical approach for bathymetric retrieval, and the accuracies and processing times were inversely related to the complexity of the models used; (2) all inversion methods provided moderately accurate retrievals of bathymetry, water column inherent optical properties, and benthic reflectance in waters less than 13 m deep with homogeneous to heterogeneous benthic/substrate covers; (3) slightly higher accuracy retrievals were obtained from locally parameterized methods; and (4) no method compared here can be considered optimal for all situations. The results provide a guide to the conditions where each approach may be used (available image and field data and processing capability). A re-analysis of these same or additional sites with satellite hyperspectral data with lower spatial and radiometric resolution, but higher temporal resolution would be instructive to establish guidelines for repeatable regional to global scale shallow water mapping approaches

Human Genotyping and An Experimental Model Reveal NPR-C as A Possible Contributor to Morbidity In Coarctation Of The Aorta

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-Cvariants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function

Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology

HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships

HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection.Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity.These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules

The pyramidalis-anterior pubic ligament-adductor longus complex (PLAC) and its role with adductor injuries: a new anatomical concept.

PURPOSE: Adductor longus injuries are complex. The conflict between views in the recent literature and various nineteenth-century anatomy books regarding symphyseal and perisymphyseal anatomy can lead to difficulties in MRI interpretation and treatment decisions. The aim of the study is to systematically investigate the pyramidalis muscle and its anatomical connections with adductor longus and rectus abdominis, to elucidate injury patterns occurring with adductor avulsions. METHODS: A layered dissection of the soft tissues of the anterior symphyseal area was performed on seven fresh-frozen male cadavers. The dimensions of the pyramidalis muscle were measured and anatomical connections with adductor longus, rectus abdominis and aponeuroses examined. RESULTS: The pyramidalis is the only abdominal muscle anterior to the pubic bone and was found bilaterally in all specimens. It arises from the pubic crest and anterior pubic ligament and attaches to the linea alba on the medial border. The proximal adductor longus attaches to the pubic crest and anterior pubic ligament. The anterior pubic ligament is also a fascial anchor point connecting the lower anterior abdominal aponeurosis and fascia lata. The rectus abdominis, however, is not attached to the adductor longus; its lateral tendon attaches to the cranial border of the pubis; and its slender internal tendon attaches inferiorly to the symphysis with fascia lata and gracilis. CONCLUSION: The study demonstrates a strong direct connection between the pyramidalis muscle and adductor longus tendon via the anterior pubic ligament, and it introduces the new anatomical concept of the pyramidalis-anterior pubic ligament-adductor longus complex (PLAC). Knowledge of these anatomical relationships should be employed to aid in image interpretation and treatment planning with proximal adductor avulsions. In particular, MRI imaging should be employed for all proximal adductor longus avulsions to assess the integrity of the PLAC

Efficacy, patient-reported outcomes, and safety of the anti-granulocyte macrophage colony-stimulating factor antibody otilimab (GSK3196165) in patients with rheumatoid arthritis: a randomised, phase 2b, dose-ranging study

The human monoclonal antibody otilimab inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF), a key driver in immune-mediated inflammatory conditions. We aimed to evaluate the efficacy, safety, and key patient-reported outcomes related to pain in patients with active rheumatoid arthritis receiving otilimab. Methods. This phase 2b, dose-ranging, multicentre, placebo-controlled study was done at 64 sites across 14 countries. Patients aged 18 years or older with rheumatoid arthritis who were receiving stable methotrexate were randomly assigned (1:1:1:1:1:1) to subcutaneous placebo or otilimab 22·5 mg, 45 mg, 90 mg, 135 mg, or 180 mg, plus methotrexate, once weekly for 5 weeks, then every other week until week 50. The randomisation schedule was generated by the sponsor, and patients were assigned to treatment by interactive response technology. Randomisation was blocked (block size of six) but was not stratified. Investigators, patients, and the sponsor were blinded to treatment. An unblinded administrator prepared and administered the study drug. The primary endpoint was the proportion of patients who achieved disease activity score for 28 joints with C-reactive protein (DAS28-CRP) 3·2 (week 24) escaped to otilimab 180 mg. Patients who escaped were treated as non-responders in their original assigned group. Safety endpoints were incidence of adverse events and serious adverse events, infections, and pulmonary events. Efficacy and safety outcomes were assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, NCT02504671. Findings.Between July 23, 2015, and Dec 29, 2017, 222 patients were randomly assigned (37 to each group). 86 (49%) of 175 escaped to otilimab 180 mg at week 12 and 57 (69%) of 83 at week 24. At week 24, the proportion of patients with DAS28-CRP <2·6 was two (5%) of 37 in the otilimab 22·5 mg group, six (16%) of 37 in the 45 mg group, seven (19%) of 37 in the 90 mg group, five (14%) of 37 in the 135 mg group, five (14%) of 37 in the 180 mg, and one (3%) of 37 in the placebo group. The largest difference was achieved with otilimab 90 mg (16·2%; odds ratio [OR] 8·39, 95% CI 0·98–72·14; p=0·053). Adverse events were reported pre-escape in 19–24 (51–65%) patients and post escape in 10–17 (40–61%) patients across otilimab dose groups and in 18 (49%) of 37 and 22 (67%) of 33 in the placebo group. The most common adverse event was nasopharyngitis: 3–9 (8–24%) in otilimab groups and one (3%) in the placebo group pre-escape and 1–3 (4–10%) in otilimab groups and seven (21%) in the placebo group post escape. Pre-escape serious adverse events were foot fracture (otilimab 45 mg); arthralgia, myocardial infarction, dizziness (otilimab 90 mg); oesophageal spasm, acute pyelonephritis (otilimab 22·5 mg), and uterine leiomyoma (otilimab 135 mg). Post-escape serious adverse events were ankle fracture (placebo) and rheumatoid arthritis (otilimab 135 mg). There were no deaths or pulmonary events of clinical concern, and rates of serious infection were low. Interpretation. Otilimab plus methotrexate was well tolerated and, despite not achieving the primary endpoint of DAS28-CRP remission, there were improvements compared with placebo in disease activity scores. Of note, patients reported significant improvement in pain and physical function, supporting further clinical development of otilimab in rheumatoid arthritis