Engineered transcription-associated Cas9 targeting in eukaryotic cells.

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT).

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind Ca2 calcium channel C-termini in vitro.

PDZ domain mediated interactions with voltage-gated calcium (Ca) channel C-termini play important roles in localizing membrane Ca signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca2.2 in mammals.

Early Penile and Donor Site Sensory Outcomes After Innervated Radial Forearm Free Flap Phalloplasty: A Pilot Prospective Study.

Background: Radial forearm free flap phalloplasty (RFFF) is a set of complex reconstructive procedures aimed at creating an aesthetic and functional penis in transgender patients. Sensory recovery in the neophallus and donor site is crucial for optimizing outcomes, but the few prior studies that exist assess neophallus sensation at limited locations and time points. The purpose of this study was to prospectively quantify sensory outcomes in the neophallus and donor site following RFFF phalloplasty.

The anterior-based muscle-sparing approach is cost-effective when compared with posterolateral and direct anterior approaches for total hip arthroplasty.

Purpose: Research relating to Total Hip Arthroplasty (THA) has found the anterior-based muscle-sparing (ABMS) approach improves both intraoperative and postoperative outcomes when compared to other THA approaches. This study compares the costs and outcomes of the ABMS approach and standard of care (SOC) to determine the relative cost-effectiveness.

Methods: A decision-analytic model was utilized to estimate intraoperative outcomes (i.

A familial, telomere-to-telomere reference for human mutation and recombination from a four-generation pedigree.

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 single-nucleotide variants (SNVs), 7.

Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer.

Although only a fraction of CTCF motifs are bound in any cell type, and approximately half of the occupied sites overlap cohesin, the mechanisms underlying cell-type specific attachment and ability to function as a chromatin organizer remain unknown. To investigate the relationship between CTCF and chromatin we applied a combination of imaging, structural and molecular approaches, using a series of brain and cancer associated CTCF mutations that act as CTCF perturbations. We demonstrate that binding and the functional impact of WT and mutant CTCF depend not only on the unique properties of each protein, but also on the genomic context of bound sites.

Mint/X11 PDZ domains from non-bilaterian animals recognize and bind Ca 2 calcium channel C-termini .

PDZ domain mediated interactions with voltage-gated calcium (Ca ) channel C-termini play important roles in localizing membrane Ca signaling. The first such interaction was described between the scaffolding protein Mint-1 and Ca 2.2 in mammals.

Binding domain mutations provide insight into CTCF's relationship with chromatin and its contribution to gene regulation.

Here we used a series of CTCF mutations to explore CTCF's relationship with chromatin and its contribution to gene regulation. CTCF's impact depends on the genomic context of bound sites and the unique binding properties of WT and mutant CTCF proteins. Specifically, CTCF's signal strength is linked to changes in accessibility, and the ability to block cohesin is linked to its binding stability.

Effects of parental age and polymer composition on short tandem repeat mutation rates.

Short tandem repeats (STRs) are hotspots of genomic variability in the human germline because of their high mutation rates, which have long been attributed largely to polymerase slippage during DNA replication. This model suggests that STR mutation rates should scale linearly with a father's age, as progenitor cells continually divide after puberty. In contrast, it suggests that STR mutation rates should not scale with a mother's age at her child's conception, since oocytes spend a mother's reproductive years arrested in meiosis II and undergo a fixed number of cell divisions that are independent of the age at ovulation.

Preparing for Death While Investing in Life: A Narrative Inquiry and Case Report of Home-Based Paediatric Palliative, End-of-Life, and After-Death Care.

Paediatric palliative care is pivotal for addressing the complex needs of children with incurable diseases and their families. While home-based care offers a familiar and supportive environment, delivering comprehensive services in this context is challenging. The existing literature on home-based palliative care lacks detailed guidance for its organization and implementation.

Manipulating the 3D organization of the largest synthetic yeast chromosome.

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction.

Engineered transcription-associated Cas9 targeting in eukaryotic cells.

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT).

Sociodemographics of Patient Populations Undergoing Gender-Affirming Surgery: A Systematic Review of All Cohort Studies.

Importance: Sociodemographic and health characteristics of patients undergoing gender-affirming surgery (GAS) are currently unknown. Understanding these patient characteristics is vital to optimizing patient-centered care for transgender patients.

Objective: To determine sociodemographic characteristics for the transgender population undergoing GAS.

Oncogenic inspiration for programmable activators.

The discovery of regulatory domains has been limited to the investigation of transcription factors and homologous protein sequences. In this issue of , motivated by an oncogenic protein fusion, Tak et al. direct the regulatory potential of a nontraditional effector domain to novel genomic loci with fusions to programmable DNA-binding domains.

A universal deep-learning model for zinc finger design enables transcription factor reprogramming.

CysHis zinc finger (ZF) domains engineered to bind specific target sequences in the genome provide an effective strategy for programmable regulation of gene expression, with many potential therapeutic applications. However, the structurally intricate engagement of ZF domains with DNA has made their design challenging. Here we describe the screening of 49 billion protein-DNA interactions and the development of a deep-learning model, ZFDesign, that solves ZF design for any genomic target.

Building an Academic Transgender Medicine Center of Excellence: The 5-Year Johns Hopkins Experience.

Gender-affirming care for transgender and gender diverse (TGD) individuals is a multidisciplinary endeavor that requires organized efforts of many specialized practitioners. TGD individuals experience many health care barriers, including the scarcity of multidisciplinary teams formed to coordinate and deliver complex care in an efficient and affirming way. The Johns Hopkins Center for Transgender Health was founded in 2017 with the mission of decreasing health disparities and improving the health of the TGD community.

Fifteen years of shared care for paediatric oncology, haematology and palliative patients across Queensland: The role of Regional Case Managers.

Objective: A shared care model was implemented in 2006 in Queensland to facilitate paediatric oncology, haematology and palliative care patients receiving care as close to home as possible. Following initial diagnosis, care planning and treatment at the tertiary children's hospital, appropriate local care was coordinated by Regional Case Managers (RCMs) established at each of 10 Shared Care Units (SCUs). This enabled safe and quality regional care supported by a statewide network providing clinical governance and education.

Tandem Walk in Simulated Martian Gravity and Visual Environment.

Astronauts returning from long-duration spaceflights experience visual-vestibular conflicts that causes motion sickness, perceptions that the environment is moving when it is not, and problems with walking and other functional tasks. To evaluate whether astronauts will have similar decrements after they land on Mars following exposure to weightlessness, participants were held by a device that offloads their weight, first entirely (0 G), and then partially (0.38 G) or not at all (1 G).

Addition of Allograft and Acromioclavicular Cerclage Improves Outcomes of Arthroscopic-Assisted Reconstruction of Acromioclavicular Separation With a Single Coracoclavicular Tunnel.

Objective The purpose of this study was to prospectively evaluate the functional outcome and complications of unstable acromioclavicular (AC) joint separations repaired with a single coracoclavicular tunnel utilizing an arthroscopic-assisted curved button technique. Methods Thirty-five patients with a minimum of 12 months follow-up underwent arthroscopic-assisted AC joint reconstruction with suspensory button and 2 mm suture tape fixation using 3 mm tunnels. Functional outcome scores were analyzed preoperatively and at final follow-up with all complications noted.

Location of end-of-life care of children with cancer: A systematic review of parent experiences.

Objective: To synthesize existing qualitative research exploring the experiences of parents caring for children with cancer during the end-of-life phase, and the factors that influence parental decision-making when choosing the location of end-of-life care and death for their child.

Results: This review included 15 studies of 460 parents of 333 children and adolescents who died from progressive cancer. Where reported, the majority (58%) of children died at home or in a hospital (39%), with only a small fraction dying in a hospice.

Familial long-read sequencing increases yield of de novo mutations.

Studies of de novo mutation (DNM) have typically excluded some of the most repetitive and complex regions of the genome because these regions cannot be unambiguously mapped with short-read sequencing data. To better understand the genome-wide pattern of DNM, we generated long-read sequence data from an autism parent-child quad with an affected female where no pathogenic variant had been discovered in short-read Illumina sequence data. We deeply sequenced all four individuals by using three sequencing platforms (Illumina, Oxford Nanopore, and Pacific Biosciences) and three complementary technologies (Strand-seq, optical mapping, and 10X Genomics).

Engineered dual selection for directed evolution of SpCas9 PAM specificity.

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs.

Outcomes Following Double-Row and Medial Double-Pulley Rotator Cuff Repair.

Multiple arthroscopic double-row repair techniques have been developed to treat full-thickness rotator cuff tears. However, healing rates and functional outcomes vary among these repair methods. A suture tape and medial double-pulley (STDP) technique that incorporated 2-mm suture tape for a knotless double-row with a broad double-mattress suture medially was developed as an alternative method to other double-row repair techniques.