HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a.

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing.

Poly-γ-glutamylation of biomolecules.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

BrAIST-Calc: Prediction of Individualized Benefit From Bracing for Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Prospective multicenter study data were used for model derivation and externally validated using retrospective cohort data. OBJECTIVE: Derive and validate a prognostic model of benefit from bracing for adolescent idiopathic scoliosis (AIS). SUMMARY OF BACKGROUND DATA: The Bracing in Adolescent Idiopathic Scoliosis Trial (BrAIST) demonstrated the superiority of bracing over observation to prevent curve progression to the surgical threshold; 42% of untreated subjects had a good outcome, and 28% progressed to the surgical threshold despite bracing, likely due to poor adherence. To avoid over-treatment and to promote patient goal setting and adherence, bracing decisions (who and how much) should be based on physician and patient discussions informed by individual-level data from high-quality predictive models. MATERIALS AND METHODS: Logistic regression was used to predict curve progression to <45° at skeletal maturity (good prognosis) in 269 BrAIST subjects who were observed or braced. Predictors included age, sex, body mass index, Risser stage, Cobb angle, curve pattern, and treatment characteristics (hours of brace wear and in-brace correction). Internal and external validity were evaluated using jackknifed samples of the BrAIST data set and an independent cohort (n=299) through estimates of discrimination and calibration. RESULTS: The final model included age, sex, body mass index, Risser stage, Cobb angle, and hours of brace wear per day. The model demonstrated strong discrimination ( c -statistics 0.83-0.87) and calibration in all data sets. Classifying patients as low risk (high probability of a good prognosis) at the probability cut point of 70% resulted in a specificity of 92% and a positive predictive value of 89%. CONCLUSION: This externally validated model can be used by clinicians and families to make informed, individualized decisions about when and how much to brace to avoid progression to surgery. If widely adopted, this model could decrease overbracing of AIS, improve adherence, and, most importantly, decrease the likelihood of spinal fusion in this population.

DNA on the move: mechanisms, functions and applications of transposable elements.

Transposons are mobile genetic elements that have invaded all domains of life by moving between and within their host genomes. Due to their mobility (or transposition), transposons facilitate horizontal gene transfer in bacteria and foster the evolution of new molecular functions in prokaryotes and eukaryotes. As transposition can lead to detrimental genomic rearrangements, organisms have evolved a multitude of molecular strategies to control transposons, including genome defense mechanisms provided by CRISPR-Cas systems. Apart from their biological impacts on genomes, DNA transposons have been leveraged as efficient gene insertion vectors in basic research, transgenesis and gene therapy. However, the close to random insertion profile of transposon-based tools limits their programmability and safety. Despite recent advances brought by the development of CRISPR-associated genome editing nucleases, a strategy for efficient insertion of large, multi-kilobase transgenes at user-defined genomic sites is currently challenging. The discovery and experimental characterization of bacterial CRISPR-associated transposons (CASTs) led to the attractive hypothesis that these systems could be repurposed as programmable, site-specific gene integration technologies. Here, we provide a broad overview of the molecular mechanisms underpinning DNA transposition and of its biological and technological impact. The second focus of the article is to describe recent mechanistic and functional analyses of CAST transposition. Finally, current challenges and desired future advances of CAST-based genome engineering applications are briefly discussed.

A neonate with aseptic cutaneous necrosis related to the migration of the proximal central venous catheter infusion port.

We report a case of aseptic cutaneous necrosis from extravasation of calcium chloride at the proximal port of a central venous catheter (CVC). A right internal jugular CVC was placed with ultrasound guidance using contemporary guidelines for size and insertion site. Catheter migration occurred concurrent with development of postoperative anasarca. Four days later, leakage of infusate with skin necrosis was noted at the insertion site. Despite initial proper positioning, catheter ports can migrate out of intravascular structures due to postprocedural subcutaneous edema. Intravascular confirmation should be performed regularly for infants with localized or generalized edema.

[Recycling of Disposable Surgical Instruments - Is It Worth It?] / Recycling von chirurgischen Einweginstrumenten ­ lohnt sich das?

The German healthcare sector is responsible for 5.2% of the country's greenhouse gas emissions. One contributing factor is the enormous amount of waste generated daily in German hospitals, making them the fifth largest waste producer in Germany. Despite the potential for recycling, a significant portion of hospital waste is incinerated, as mandated by current regulations. This results in high levels of noxious CO2 emissions and the loss of valuable resources. The goal of this project was to demonstrate the feasibility of recycling complex, contaminated disposable surgical instruments.The study included frequently used disposable surgical instruments that could potentially be recycled as electronic waste. The instruments were wipe-disinfected and sterilised internally within the hospital. After sterilisation, the devices could be classified as electronic waste in consultation with the environmental authorities and then machine-recycled externally by a waste disposal company. Sorting machines shredded and separated the instruments into individual fractions of cables, plastics, different metals, and circuit boards, which were further processed into secondary raw materials.In the first six months (09/2022-03/2023), 239 kg of material were recycled instead of being incinerated. This resulted in a reduction of 545 kg CO2e. The metal content was estimated as 50% of the total weight; 30% were recyclable plastics, resulting in an 80% recycling rate. The ongoing recycling costs were 1.90 €/kg after deducting revenues. Thus, recycling in this model was approximately 3.9 times as expensive as incineration. A survey of the operating theatre personnel found high satisfaction with the recycling project and a minimal additional workload of less than five minutes.We demonstrated that recycling of contaminated disposable surgical instruments is possible in coordination with government authorities. This approach avoids waste incineration and leads to a reduction in CO2-equivalent emissions. However, the higher costs of recycling and the requirement for in-house decontamination pose limitations on the implementation of such projects. To address this, it is necessary for lawmakers to reconsider current regulations and involve manufacturers in recycling costs to fully exploit the enormous recycling potential.

Chance Fracture Pattern Presenting in Proximal Junctional Failure.

INTRODUCTION: We present a case series of proximal junctional failure due to a Chance-type fracture. METHODS: This is a retrospective review of patients who developed proximal junctional kyphosis because of Chance-type proximal junctional failure after spinal fusion for adult spinal deformity. RESULTS: Fifteen patients were identified (4M:11F). The average age was 61.4 years (range, 39 to 77). The mean time to fracture identification was 25.4 days (range, 3 to 65). The average number of levels instrumented was 6.7 (range, 2 to 17). No patients had antecedent trauma before fracture onset. In 67% of cases with a lumbar upper instrumented vertebra (UIV), there was overcorrection of lumbar lordosis (LL) and/or lower LL. The five cases with a lower thoracic UIV had undergone notable correction of preoperative thoracolumbar junction kyphosis. 14 of 15 patients were treated with extension of fusion. Pedicle screws at the fracture level were salvaged by changing to an anatomic trajectory. CONCLUSION: Continued pain at 6 to 12 weeks with radiographs showing an increased proximal junctional angle and cephalocaudal pedicle widening at the UIV should raise suspicion for this unique fracture pattern. A CT scan is recommended. Low bone density, LL and/or lower LL overcorrection, and selection of lower thoracic UIV in the setting of notable thoracolumbar junction correction may contribute to fracture risk.

Are Grit Scale Scores or Connor-Davidson Resilience Scale Scores Correlated with Career Achievements Among Physical Therapy Program Graduates?

AIMS: This study examined how the qualities of grit and resilience correlated with career achievements in physical therapists. The purpose of this study was to determine if select career achievements were correlated with 1) Original Grit Scale (Grit-O) scores, 2) Short Grit Scale (Grit-S) scores, or 3) Connor-Davidson Resilience Scale (CD-RISC) scores among Mayo Clinic School of Health Sciences Physical Therapy Program graduates from 2000-2018. METHODS: Cross-sectional research design. Participants were 212 graduates between 2000 and 2018, inclusive. Participants completed the Grit-O, Grit-S, and CD-RISC scales and reported career achievements. Descriptive statistics were used to summarize the demographics, career achievements, grit, and resilience of the subjects. Point biserial and partial correlations were used to examine associations between Grit-O and Grit-S subscales, CD-RISC scores, and career achievements. RESULTS: When controlled for gender and time since graduation, there were significant positive relationships between Grit-O Perseverance of Effort and 1) publication in a peer-reviewed journal and 2) attainment of an additional degree. Biological males were significantly more likely to have reported certain career achievements. CONCLUSION: Few of the expected relationships were found, possibly due to a lack of true relationships, a homogeneous population, ceiling effect, or inaccurate self-reports.

Phonon-mediated room-temperature quantum Hall transport in graphene.

The quantum Hall (QH) effect in two-dimensional electron systems (2DESs) is conventionally observed at liquid-helium temperatures, where lattice vibrations are strongly suppressed and bulk carrier scattering is dominated by disorder. However, due to large Landau level (LL) separation (~2000 K at B = 30 T), graphene can support the QH effect up to room temperature (RT), concomitant with a non-negligible population of acoustic phonons with a wave-vector commensurate to the inverse electronic magnetic length. Here, we demonstrate that graphene encapsulated in hexagonal boron nitride (hBN) realizes a novel transport regime, where dissipation in the QH phase is governed predominantly by electron-phonon scattering. Investigating thermally-activated transport at filling factor 2 up to RT in an ensemble of back-gated devices, we show that the high B-field behaviour correlates with their zero B-field transport mobility. By this means, we extend the well-accepted notion of phonon-limited resistivity in ultra-clean graphene to a hitherto unexplored high-field realm.

Factors Associated With Vaccine-Induced T-Cell Immune Responses Against Severe Acute Respiratory Syndrome Coronavirus 2 in Kidney Transplant Recipients.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important prophylactic measure in kidney transplant recipients (KTRs), but the immune response is often impaired. Here, we examined the T-cell immune response against SARS-CoV-2 in 148 KTRs after 3 or 4 vaccine doses, including 35 KTRs with subsequent SARS-CoV-2 infection. The frequency of spike-specific T cells was lower in KTRs than in immunocompetent controls and was correlated with the level of spike-specific antibodies. Positive predictors for detection of vaccine-induced T cells were detection of spike-specific antibodies, heterologous immunization with messenger RNA and a vector vaccine, and longer time after transplantation. In vaccinated KTRs with subsequent SARS-CoV-2 infection, the T-cell response was greatly enhanced and was significantly higher than in vaccinated KTRs without SARS-CoV-2 infection. Overall, the data show a correlation between impaired humoral and T-cell immunity to SARS-CoV-2 vaccination and provide evidence for greater robustness of hybrid immunity in KTRs.

FLASHlab@PITZ: New R&D platform with unique capabilities for electron FLASH and VHEE radiation therapy and radiation biology under preparation at PITZ.

At the Photo Injector Test facility at DESY in Zeuthen (PITZ), an R&D platform for electron FLASH and very high energy electron radiation therapy and radiation biology is being prepared (FLASHlab@PITZ). The beam parameters available at PITZ are worldwide unique. They are based on experiences from 20 + years of developing high brightness beam sources and an ultra-intensive THz light source demonstrator for ps scale electron bunches with up to 5 nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms length, currently 22 MeV (upgrade to 250 MeV planned). Individual bunches can provide peak dose rates up to 1014 Gy/s, and 10 Gy can be delivered within picoseconds. Upon demand, each bunch of the bunch train can be guided to a different transverse location, so that either a "painting" with micro beams (comparable to pencil beam scanning in proton therapy) or a cumulative increase of absorbed dose, using a wide beam distribution, can be realized at the tumor. Full tumor treatment can hence be completed within 1 ms, mitigating organ movement issues. With extremely flexible beam manipulation capabilities, FLASHlab@PITZ will cover the current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexploited short treatment times and high dose rates. A summary of the plans for FLASHlab@PITZ and the status of its realization will be presented.

Structural basis for the assembly of the type V CRISPR-associated transposon complex.

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.

Intramedullary Nail and Plate Combination Technique for Peri-Implant Both-Bone Forearm Fractures.

A 35-year-old female patient with cerebellar ataxia presented with a right periprosthetic both-bone forearm fracture after a ground-level fall. Her surgical history was significant for multiple both-bone forearm fractures treated by open reduction and internal fixation. Subsequent treatment with a combination of intramedullary nailing and plate fixation for each bone provided successful fracture union while allowing immediate return to weight-bearing and range of motion. This case report demonstrates that intramedullary nailing and plate fixation of both-bone forearm fractures provides complete protection of the radius and ulna in recurrent, peri-implant both-bone forearm fractures. This technique is a valuable treatment option in the setting of a patient at risk for recurrent injury of the forearm.

Supratherapeutic Antibiotic Levels and Acute Kidney Injury from Absorption of Topical Antibiotics: A Case Report.

CASE: A 96-year-old woman with no baseline renal dysfunction presented with a distal femoral shaft fracture after a ground-level fall. Treatment was with a retrograde intramedullary nail and included placement of topical antibiotics. Postoperatively, she developed acute kidney injury and was found to have supratherapeutic antibiotic levels. CONCLUSION: This case report demonstrates the risk of clinically relevant systemic absorption along with associated downstream end organ damage with the use of topical antibiotics in certain circumstances. We present this case as an illustration of a rare hazard associated with topical antibiotic use.

Immune response to third SARS-CoV-2 vaccination in seronegative kidney transplant recipients: Possible improvement by mycophenolate mofetil reduction.

Modification of vaccination strategies is necessary to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). This multicenter observational study analyzed the effects of the third SARS-CoV-2 vaccination in previously seronegative KTRs with the focus on temporary mycophenolate mofetil (MMF) dose reduction within propensity matched KTRs. 56 out of 174 (32%) previously seronegative KTRs became seropositive after the third vaccination with only three KTRs developing neutralizing antibodies against the omicron variant. Multivariate logistic regression revealed that initial antibody levels, graft function, time after transplantation and MMF trough levels had an influence on seroconversion (P < .05). After controlling for confounders, the effect of MMF dose reduction before the third vaccination was calculated using propensity score matching. KTRs with a dose reduction of ≥33% showed a significant decrease in MMF trough levels to 1.8 (1.2-2.5) µg/ml and were more likely to seroconvert than matched controls (P = .02). Therefore, a MMF dose reduction of 33% or more before vaccination is a promising approach to improve success of SARS-CoV-2 vaccination in KTRs.

Bacterial Cellulose-Adaptation of a Nature-Identical Material to the Needs of Advanced Chronic Wound Care.

Modern wound treatment calls for hydroactive dressings. Among the variety of materials that have entered the field of wound care in recent years, the carbohydrate polymer bacterial cellulose (BC) represents one of the most promising candidates as the biomaterial features a high moisture-loading and donation capacity, mechanical stability, moldability, and breathability. Although BC has already gained increasing relevance in the treatment of burn wounds, its potential and clinical performance for "chronic wound" indications have not yet been sufficiently investigated. This article focuses on experimental and clinical data regarding the application of BC within the indications of chronic, non-healing wounds, especially venous and diabetic ulcers. A recent clinical observation study in a chronic wound setting clearly demonstrated its wound-cleansing properties and ability to induce healing in stalling wounds. Furthermore, the material parameters of BC dressings obtained through the static cultivation of Komagataeibacter xylinus were investigated for the first time in standardized tests and compared to various advanced wound-care products. Surprisingly, a free swell absorptive capacity of a BC dressing variant containing 97% moisture was found, which was higher than that of alginate or even hydrofiber dressings. We hypothesize that the fine-structured, open porous network and the resulting capillary forces are among the main reasons for this unexpected result.

Variability in evaluation and treatment of tibial tubercle fractures among pediatric orthopedic surgeons.

The purpose of this study was to determine the variability in clinical management of tibial tubercle fractures among a group of pediatric orthopedic surgeons. Nine fellowship-trained academic pediatric orthopedic surgeons reviewed 51 anteroposterior and lateral knee radiographs with associated case age. Respondents were asked to describe each fracture using the Ogden classification (type 1-5 with A/B modifiers), desired radiographic workup, operative vs. nonoperative treatment strategy and plans for post-treatment follow-up. Fair agreement was reached when classifying the fracture type using the Ogden classification (k = 0.39; P < 0.001). Overall, surgeons had a moderate agreement on whether to treat the fractures operatively vs. nonoperatively (k = 0.51; P < 0.001). Nonoperative management was selected for 80.4% (45/56) of type 1A fractures. Respondents selected operative treatment for 75% (30/40) of type 1B, 58.3% (14/24) of type 2A, 97.4% (74/76) of type 2B, 90.7% (39/43) of type 3A, 96.3% (79/82) of type 3B, 71.9% (87/121) of type 4 and 94.1% (16/17) of type 5 fractures. Regarding operative treatment, fair/slight agreement was reached when selecting the specifics of operative treatment including surgical fixation technique (k = 0.25; P < 0.001), screw type (k = 0.26; P < 0.001), screw size (k = 0.08; P < 0.001), use of washers (k = 0.21; P < 0.001) and performing a prophylactic anterior compartment fasciotomy (k = 0.20; P < 0.001). Furthermore, surgeons had fair/moderate agreement regarding the specifics of nonoperative treatment including degree of knee extension during immobilization (k = 0.46; P < 0.001), length of immobilization (k = 0.34; P < 0.001), post-treatment weight bearing status (k = 0.30; P < 0.001) and post-treatment rehabilitation (k = 0.34; P < 0.001). Significant variability exists between surgeons when evaluating and treating pediatric tibial tubercle fractures.