Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.

Accurately quantifying the functional consequences of noncoding mosaic variants requires the pairing of DNA sequences with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic, and epigenomic profiling across targeted >100 kb loci with ∼10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent.

and expansion of CD33/HBG promoter-edited HSPCs with Mylotarg.

We developed an HSC gene therapy approach that consists of HSC mobilization and intravenous injection of HSC-tropic HDAd vectors. To achieve therapeutically relevant numbers of corrected cells, we incorporated expansion of transduced cells. We used an HDAd vector for a multiplex adenine base editing approach to (1) remove the region within CD33 that is recognized by gemtuzumab ozogamicin (GO) (Mylotarg), and (2) create therapeutic edits within the HBG1/2 promoters to reactivate γ-globin/HbF.

Auto-expansion of HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2.

We developed an hematopoietic stem cell (HSC) gene therapy approach that does not require cell transplantation. To achieve therapeutically relevant numbers of corrected cells, we constructed HSC-tropic HDAd5/35++ vectors expressing a 3' UTR truncated HMGA2 gene and a GFP reporter gene. A SB100x transposase vector mediated random integration of the tHMGA2/GFP transgene cassette.

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq.

Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules-a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with ~10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition.

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood.

A simplified G-CSF-free procedure allows for in vivo HSC gene therapy of sickle cell disease in a mouse model.

We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4β1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice.

CD46 and CD59 inhibitors enhance complement-dependent cytotoxicity of anti-CD38 monoclonal antibodies daratumumab and isatuximab in multiple myeloma and other B-cell malignancy cells.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage. Remarkable progress has been made in the treatment of MM with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab, which can kill MM cells by inducing complement-dependent cytotoxicity (CDC). We showed that the CDC efficacy of daratumumab and isatuximab is limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells.

Impact of Surgeon Case Volume on Outcomes After Reverse Total Shoulder Arthroplasty.

Introduction: Despite a rapid increase in utilization of reverse total shoulder arthroplasty (rTSA), volume-outcome studies focusing on surgeon volume are lacking. Surgeon-specific volume-outcome studies may inform policymakers and provide insight into learning curves and measures of efficiency with greater case volume.

Methods: This retrospective cohort study with longitudinal data included all rTSA cases as recorded in the Centers for Medicare & Medicaid Services Limited Data Set (2016 to 2018).

Perioperative pain protocols following surgery for adolescent idiopathic scoliosis: a snapshot of current treatments utilized by attending orthopedic surgeons.

Purpose: Perioperative management after adolescent idiopathic scoliosis (AIS) surgery varies extensively between surgeons and institutions. We devised a questionnaire to assess surgeon baseline characteristics, practice settings, and pain regimens to assess what factors contribute to perioperative pain protocols.

Methods: A multiple-choice questionnaire including 130 independent variables regarding baseline characteristics, practice environments, and pain regimen protocols was distributed to elicit information among surgeons performing AIS fusion surgery.

Implementation of a Core-Shell Design Approach for Constructing MOFs for CO Capture.

Adsorption-based capture of CO from flue gas and from air requires materials that have a high affinity for CO and can resist water molecules that competitively bind to adsorption sites. Here, we present a core-shell metal-organic framework (MOF) design strategy where the core MOF is designed to selectively adsorb CO, and the shell MOF is designed to block HO diffusion into the core. To implement and test this strategy, we used the zirconium (Zr)-based UiO MOF platform because of its relative structural rigidity and chemical stability.

Factors Associated With Amputation Following Ankle Fracture Surgery.

Patients with diabetes mellitus (DM) are at increased risk of complications following ankle fracture surgery. Previous research suggests that patients of low socioeconomic status are at increased risk of amputation following orthopedic complications. The purpose of this research was to determine if low socioeconomic status increases risk of below-knee amputation (BKA) following ankle fractures among patients with DM.

HDAd6/35++ - A new helper-dependent adenovirus vector platform for transduction of hematopoietic stem cells.

In previous studies, we achieved safe and efficient hematopoietic stem cell (HSC) transduction in mobilized mice and macaques with intravenously injected helper-dependent adenovirus HDAd5/35++ vectors. These vectors are derivatives of serotype Ad5-containing CD46-affinity enhanced Ad35 fiber knob domains. Considering the impact of anti-Ad5/HDAd5/35++ neutralizing serum antibodies present in the human population, we generated HSC-retargeted HDAd6/35++ vectors derived from serotype 6.

In vivo HSC prime editing rescues sickle cell disease in a mouse model.

Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice).

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 μg/mL (mice) and >20 μg/mL (rhesus) with half maximal inhibitory concentrations (ICs) of 1 μg/mL measured by an HIV neutralization assay.

Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors.

Desmoglein 2 (DSG2) is overexpressed in many epithelial cancers and therefore represents a target receptor for oncolytic viruses, including Ad5/3-based viruses. For most Ad serotypes, the receptor-binding fiber is composed of tail, shaft, and knob domains. Here, we investigated the role of the fiber shaft in Ad5/3 tumor transduction in vitro and in human DSG2-transgenic mice carrying human DSG2 tumors.

Designing optimal core-shell MOFs for direct air capture.

Metal-organic frameworks (MOFs), along with other novel adsorbents, are frequently proposed as candidate materials to selectively adsorb CO for carbon capture processes. However, adsorbents designed to strongly bind CO nearly always bind HO strongly (sometimes even more so). Given that water is present in significant quantities in the inlet streams of most carbon capture processes, a method that avoids HO competition for the CO binding sites would be technologically valuable.

Properties of Adenovirus Vectors with Increased Affinity to DSG2 and the Potential Benefits of Oncolytic Approaches and Gene Therapy.

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening.

In vivo base editing by a single i.v. vector injection for treatment of hemoglobinopathies.

Individuals with β-thalassemia or sickle cell disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% fetal hemoglobin (HbF) appear to be symptom free. Here, we used a nonintegrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113 A>G HPFH mutation in the γ-globin promoters in healthy CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only s.

Development of a post-mortem human specimen flow model for advanced bleeding control training.

Introduction: Prompt and effective hemorrhage control is paramount to improve survival in patients with catastrophic bleeding. In the ever-expanding field of bleeding control techniques, there is a need for a realistic training model to practice these life-saving skills. This study aimed to create a realistic perfused post-mortem human specimen (PMHS) flow model that is suitable for training various bleeding control techniques.