ABE-ultramax for high-efficiency biallelic adenine base editing in zebrafish.

Advancements in CRISPR technology, particularly the development of base editors, revolutionize genetic variant research. When combined with model organisms like zebrafish, base editors significantly accelerate and refine in vivo analysis of genetic variations. However, base editors are restricted by protospacer adjacent motif (PAM) sequences and specific editing windows, hindering their applicability to a broad spectrum of genetic variants. Additionally, base editors can introduce unintended mutations and often exhibit reduced efficiency in living organisms compared to cultured cell lines. Here, we engineer a suite of adenine base editors (ABEs) called ABE-Ultramax (Umax), demonstrating high editing efficiency and low rates of insertions and deletions (indels) in zebrafish. The ABE-Umax suite of editors includes ABEs with shifted, narrowed, or broadened editing windows, reduced bystander mutation frequency, and highly flexible PAM sequence requirements. These advancements have the potential to address previous challenges in disease modeling and advance gene therapy applications.

Emergence of slip-ideal-slip behavior in tip-links serve as force filters of sound in hearing.

Tip-links in the inner ear convey force from sound and trigger mechanotransduction. Here, we present evidence that tip-links (collectively as heterotetrameric complexes of cadherins) function as force filters during mechanotransduction. Our force-clamp experiments reveal that the tip-link complexes show slip-ideal-slip bond dynamics. At low forces, the lifetime of the tip-link complex drops monotonically, indicating slip-bond dynamics. The ideal bond, rare in nature, is seen in an intermediate force regime where the survival of the complex remains constant over a wide range. At large forces, tip-links follow a slip bond and dissociate entirely to cut-off force transmission. In contrast, the individual tip-links (heterodimers) display slip-catch-slip bonds to the applied forces. While with a phenotypic mutant, we showed the importance of the slip-catch-slip bonds in uninterrupted hearing, our coarse-grained Langevin dynamics simulations demonstrated that the slip-ideal-slip bonds emerge as a collective feature from the slip-catch-slip bonds of individual tip-links.

2-Mercaptonicotinoyl glycine prevents UV-induced skin darkening and delayed tanning in healthy subjects: A randomized controlled clinical study.

BACKGROUND: Chronic nonextreme sun exposure induces two mechanisms of skin pigmentation, causing immediate darkening and delayed tanning. A new molecule, 2-mercaptonicotinoyl glycine (2-MNG), has been shown in vitro to inhibit both immediate darkening and new melanin synthesis via covalent conjugation of the thiol group of 2-MNG to melanin precursors. OBJECTIVE: To evaluate 2-MNG in preventing both mechanisms in vivo. METHODS: In a randomized, intra-individual and controlled study, 33 subjects with melanin-rich skin were exposed to UV daylight on designated areas on the back and treated with a cosmetic formula containing 0.5% or 1% 2-MNG alone or 0.5% 2-MNG in association with lipohydroxy acid (LHA, 0.3%) plus Mexoryl-SX (MSX, 1.5%). The respective vehicles were used as controls and 4-n-butyl-resorcinol (4-n-BR, 2.5%) as a positive reference. RESULTS: 2-MNG alone significantly reduced immediate darkening and inhibited new melanin production when compared with vehicle, with higher performance at 1% than at 0.5%. 2-MNG at 0.5% in association with LHA and MSX showed significantly higher performance than 2-MNG 0.5% alone. 2-MNG at 0.5% and 1% showed significantly better performance than 4-n-BR. CONCLUSIONS: 2-MNG inhibited both UV-induced skin pigmentation mechanisms in vivo. The association of 2-MNG with LHA plus MSX showed the highest efficacy on melanin-rich skin with pigmentation induced by UV exposure.

Revisiting the significance of nano-vitamin D for food fortification and therapeutic application.

OBJECTIVE: Vitamin D (a prohormone) is an important micronutrient required by the body for skeletal homeostasis and a range of non-skeletal actions. Calcitriol, the active form of vitamin D, regulates a variety of cellular and metabolic processes through both genomic and nongenomic pathways. Often prescribed for treating rickets and osteoporosis, vitamin D deficiency can exacerbate various other medical conditions. SIGNIFICANCE, METHODS, AND RESULTS: Despite its multifunctional uses, the sensitivity of vitamin D makes formulating an efficient drug delivery system a challenging task, which is further complicated by its poor aqueous solubility. Enhancing the oral absorption of vitamin D is vital in utilizing its full efficacy. Recent developments in encapsulation and nanotechnology have shown promising results in overcoming these constraints. CONCLUSION: This review thus offers an insight to adequately comprehend the mechanistic pharmacology of vitamin D, its pathophysiological role, and justification of its medical indications, along with the benefits of utilizing nanotechnology for vitamin D delivery.

The histidine kinase NahK regulates pyocyanin production through the PQS system.

Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa. This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum-sensing (QS) systems, with a large upregulation of the Pseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

IMPACT OF FOVEAL STATUS AND TIMING OF SURGERY ON VISUAL OUTCOME IN RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To investigate the impact of surgical timing on visual acuity outcomes in retinal detachments based on the preoperative foveal status. METHODS: A retrospective multicenter cohort study was conducted. Cases were stratified into fovea-on, fovea-split, and fovea-off groups. Days to surgery was defined as the time between the preoperative examination and surgery. The main outcome measure was the final postoperative visual acuity. RESULTS: 1,675 cases were studied. More than 80% of fovea-on/fovea-split and fovea-off cases had surgery within 1 and 3 days, respectively. The mean final postoperative visual acuity did not differ significantly between the fovea-on and fovea-split groups (Snellen equivalent [SE] 20/33 ± 20/49 and 20/32 ± 20/39, P = 1.000) and did not change significantly based on days to surgery in either group. The mean final postoperative visual acuity was lowest in the fovea-off group (Snellen equivalent = 20/56 ± 20/76, P < 0.001) and was significantly lower in cases where surgery was performed after two or more days when compared with cases performed within 1 day (Snellen equivalent 20/74 ± 20/89 vs. 20/46 ± 20/63, P < 0.001). CONCLUSION: Fovea-on and fovea-split retinal detachments demonstrated comparable visual outcomes. Fovea-off RDs demonstrated worse visual outcomes, which declined further when surgery was delayed by two or more days.

Patient Blood Management and Its Role in Supporting Blood Supply.

Blood donors and voluntary blood donations are essential for ensuring the blood supply that can be maintained by good patient blood management (PBM) practices. This review article explores the role of blood donation in PBM and highlights the importance of donor screening and selection processes in different regions worldwide. The donor health questionnaires and the focused physical examination guidelines have changed in the last decade to increase donor and recipient safety. This article also discusses the status of transfusion practices, including the challenges of ensuring a safe blood supply. Significant among these are the effects of the COVID-19 pandemic on the blood supply chain and the impact of an aging donor population, especially. Promoting autologous donations and other blood conservation strategies are suggested to mitigate these issues. The role of replacement donors and the upper age limit for voluntary blood donation may be decided based on the demography and donor pool. The involvement of C-suite executives is also critical in implementing and running a successful PBM program. The review highlights how these different aspects of blood donation are integral to a successful PBM program and the safety of patients who receive blood transfusions.

Evaluating the association of biallelic OGDHL variants with significant phenotypic heterogeneity.

BACKGROUND: Biallelic variants in OGDHL, encoding part of the α-ketoglutarate dehydrogenase complex, have been associated with highly heterogeneous neurological and neurodevelopmental disorders. However, the validity of this association remains to be confirmed. A second OGDHL patient cohort was recruited to carefully assess the gene-disease relationship. METHODS: Using an unbiased genotype-first approach, we screened large, multiethnic aggregated sequencing datasets worldwide for biallelic OGDHL variants. We used CRISPR/Cas9 to generate zebrafish knockouts of ogdhl, ogdh paralogs, and dhtkd1 to investigate functional relationships and impact during development. Functional complementation with patient variant transcripts was conducted to systematically assess protein functionality as a readout for pathogenicity. RESULTS: A cohort of 14 individuals from 12 unrelated families exhibited highly variable clinical phenotypes, with the majority of them presenting at least one additional variant, potentially accounting for a blended phenotype and complicating phenotypic understanding. We also uncovered extreme clinical heterogeneity and high allele frequencies, occasionally incompatible with a fully penetrant recessive disorder. Human cDNA of previously described and new variants were tested in an ogdhl zebrafish knockout model, adding functional evidence for variant reclassification. We disclosed evidence of hypomorphic alleles as well as a loss-of-function variant without deleterious effects in zebrafish variant testing also showing discordant familial segregation, challenging the relationship of OGDHL as a conventional Mendelian gene. Going further, we uncovered evidence for a complex compensatory relationship among OGDH, OGDHL, and DHTKD1 isoenzymes that are associated with neurodevelopmental disorders and exhibit complex transcriptional compensation patterns with partial functional redundancy. CONCLUSIONS: Based on the results of genetic, clinical, and functional studies, we formed three hypotheses in which to frame observations: biallelic OGDHL variants lead to a highly variable monogenic disorder, variants in OGDHL are following a complex pattern of inheritance, or they may not be causative at all. Our study further highlights the continuing challenges of assessing the validity of reported disease-gene associations and effects of variants identified in these genes. This is particularly more complicated in making genetic diagnoses based on identification of variants in genes presenting a highly heterogenous phenotype such as "OGDHL-related disorders".

Enhancing Surgical Outcomes: A Critical Review of Antibiotic Prophylaxis in Orthopedic Surgery.

The postoperative burden remains significant due to the possibility of prolonged hospitalization, escalated healthcare costs, and patient distress caused by postorthopedic surgical site infections (SSIs). Orthopedic surgery is likewise faced with a significant challenge posed by these conditions. A positive association has been observed between the presence of postorthopedic SSIs and heightened susceptibility to adverse health outcomes, along with elevated rates of morbidity and mortality. Systemic antibiotic prophylaxis (SAP) reduces the risk of acquiring an SSI. Closed fractures, open fractures, arthroplasty, and percutaneous fixation each possess distinct attributes that impact the data and antimicrobial therapy. When implementing SAP, it is crucial to strike a delicate equilibrium between maintaining effective antibiotic stewardship protocols and preventing the occurrence of SSIs. This practice effectively prevents both the incidence of negative consequences and the emergence of antibiotic resistance. The objective of this study was to examine the existing literature on the use of surgical antibiotic prophylaxis in orthopedic surgery and explore the potential consequences associated with the inappropriate administration of antibiotics.

Design and Development of a Topical Nanogel Formulation Comprising of a Unani Medicinal Agent for the Management of Pain.

The oil of the Unani medicinal herb Baboona (Matricaria chamomilla) has shown potential in the management of pain. However, predicaments such as poor skin penetration, skin sensitization, liable to degradation, and volatile nature restrict its use. Therefore, our group for the first time has developed a carrier-based delivery system to facilitate the direct application of chamomile oil to the forehead. The developed nanogel was characterized for physical parameters such as compatibility, TEM, and stability studies. Further, it was also evaluated for pH, viscosity, spread ability, and extrudability, as well as through texture analyses, in vitro studies, and skin irritation tests. The formulation was successfully developed with all the necessary attributes. The in vitro studies revealed the enhanced skin penetration of chamomile oil nanogel. The in vivo studies were also performed in chemically induced pain models, mimicking migraine. The studies show significant improvement of the pain threshold for chamomile nanogel when compared to the positive control group and the results were comparable to marketed diclofenac formulations. Finally, the encapsulation into nanogel reduced the skin irritation property. The nanogel formulation showed promising effects in the pain management of migraine.

Red Blood Cell Transfusion: 2023 AABB International Guidelines.

Importance: Red blood cell transfusion is a common medical intervention with benefits and harms. Objective: To provide recommendations for use of red blood cell transfusion in adults and children. Evidence Review: Standards for trustworthy guidelines were followed, including using Grading of Recommendations Assessment, Development and Evaluation methods, managing conflicts of interest, and making values and preferences explicit. Evidence from systematic reviews of randomized controlled trials was reviewed. Findings: For adults, 45 randomized controlled trials with 20 599 participants compared restrictive hemoglobin-based transfusion thresholds, typically 7 to 8 g/dL, with liberal transfusion thresholds of 9 to 10 g/dL. For pediatric patients, 7 randomized controlled trials with 2730 participants compared a variety of restrictive and liberal transfusion thresholds. For most patient populations, results provided moderate quality evidence that restrictive transfusion thresholds did not adversely affect patient-important outcomes. Recommendation 1: for hospitalized adult patients who are hemodynamically stable, the international panel recommends a restrictive transfusion strategy considering transfusion when the hemoglobin concentration is less than 7 g/dL (strong recommendation, moderate certainty evidence). In accordance with the restrictive strategy threshold used in most trials, clinicians may choose a threshold of 7.5 g/dL for patients undergoing cardiac surgery and 8 g/dL for those undergoing orthopedic surgery or those with preexisting cardiovascular disease. Recommendation 2: for hospitalized adult patients with hematologic and oncologic disorders, the panel suggests a restrictive transfusion strategy considering transfusion when the hemoglobin concentration is less than 7 g/dL (conditional recommendations, low certainty evidence). Recommendation 3: for critically ill children and those at risk of critical illness who are hemodynamically stable and without a hemoglobinopathy, cyanotic cardiac condition, or severe hypoxemia, the international panel recommends a restrictive transfusion strategy considering transfusion when the hemoglobin concentration is less than 7 g/dL (strong recommendation, moderate certainty evidence). Recommendation 4: for hemodynamically stable children with congenital heart disease, the international panel suggests a transfusion threshold that is based on the cardiac abnormality and stage of surgical repair: 7 g/dL (biventricular repair), 9 g/dL (single-ventricle palliation), or 7 to 9 g/dL (uncorrected congenital heart disease) (conditional recommendation, low certainty evidence). Conclusions and Relevance: It is good practice to consider overall clinical context and alternative therapies to transfusion when making transfusion decisions about an individual patient.

A Prospective Comparison of Octyl-2-Cyanoacrylate and Suture in Facial Wounds.

The study aimed to evaluate the efficacy of octyl-2-cyanoacrylate as well as sutures as a wound closure material. Whether octyl-2-cyanoacrylate can be used as an alternative to suture in the closure of facial wounds. Out of total of 19 wounds, 11 wounds were closed with sutures and tissue adhesive both, out of rest 8 wounds, 4 wounds were closed using sutures alone and in 4 wounds, closure was done with tissue adhesive alone. Therefore, a total of 15 facial wounds were divided into two groups: Group-I Cases in whom 3-0 Black silk suture was used for closure of the wound and Group-II Cases in whom Dermabond (Octyl-2-Cyanoacrylate) was used for closure of the wound. Results showed that Octyl-2-cyanoacrylate offered the benefit of decreased procedure time with less pain, no need for its removal, and better cosmetic outcome compared to sutures.

Dual-NIR wavelength (pulsed 810 nm and superpulsed 904 nm lasers) photobiomodulation therapy synergistically augments full-thickness burn wound healing: A non-invasive approach.

A thermal burn is the most frequent, distressing form of trauma. Globally, there is a critical necessity to explore novel therapeutic strategies for burn wound care. Combination therapy has marked therapeutic efficacy in positively regulating various phases of wound repair. Photobiomodulation (PBM) is a biophysical, non-thermal therapeutic healing modality to treat chronic non-healing wounds. It hypothesized that PBM using combined NIR wavelengths may absorb through different cellular photoacceptors with varying degrees of tissue penetration, which can potentially regulate the pace of healing. Therefore, the current study investigates the efficacy of dual-NIR wavelength treatment employing pulsed 810 nm and superpulsed 904 nm lasers PBM on transdermal burn repair in rats and unveils the associated molecular mechanistic insights. Rats were randomized into five groups: uninjured skin, burn control (sham-exposed), standalone treatment with pulsed 810 nm laser, superpulsed 904 nm laser, and dual combination groups. The present findings revealed that PBM with dual-NIR wavelength synergistically augmented burn wound healing compared to control and standalone treatments. The efficacy of combined treatment was exhibited by significantly enhanced wound area contraction (α-smooth muscle actin), proliferation (PCNA, cytokeratin-14, TGF-ß2), angiogenesis (HIF-1α, CD31), ECM accumulation/ organization (collagen type 3, fibronectin), dermal hydration (AQP3), calcium homeostasis (TRPV3, calmodulin), and bioenergetics activation (CCO, AMPK-α, ATP). Collectively, PBM with dual-NIR wavelength (pulsed/ superpulsed-mode) treatment accelerates full-thickness burn wound healing, which could be used as a non-invasive translational approach in clinical significance in conjunction with existing burn wound care management.

How Not To Fix a Tibial Fracture: A Case Report on Treatment By a Quack.

Quackery in the orthopaedic profession has existed for quite a long time. Due to the shortage of orthopaedic healthcare staff in public hospitals and the high costs in private facilities, members of disadvantaged communities turn to unlicensed and unskilled practitioners (quacks). The main factors responsible for the increased number of quacks performing orthopaedic treatment are illiteracy, high treatment cost, mismatch in the orthopaedic surgeon-to-population ratio, especially in rural areas, and the absence of any form of health insurance. Moreover, their easy availability and offer of low-cost treatment draw innocent and illiterate patients to them, even though these quacks perform orthopaedic treatment in the most unhygienic, unsterilized, and unconventional manner. The government should intervene and take measures to make orthopaedic treatment more affordable and accessible, especially to the rural population.

Copper-catalysed dehydrogenative self-coupling/cyclization of 5-aminopyrazoles: synthesis and photophysical study of pyridazines.

An interesting self-coupling/cyclization of 5-aminopyrazoles is revealed, which provides a variety of pyridazine cores in reasonable yields. In this reaction, C(sp2)-C(sp2) and N-N bond formation occurs simultaneously in one reaction vessel. The photophysical properties of the synthesized compounds were also studied and some of them exhibited fluorescence properties with good quantum yields. A radical mediated reaction mechanism is proposed with the help of control experiments.

Development and Antibacterial Investigation of Linezolid-Loaded SPIONs and HPLC Method Development for Quantitative Analysis of Linezolid.

BACKGROUND: Linezolid (LNZ) is extremely prone to resistance. The development of resistance to LNZ should be taken into consideration when selecting this drug as a therapeutic option. It is well established that reactive oxygen species (ROS) generated by iron oxide nanoparticles (MNPs) could kill the infecting bacteria. So, we hypothesized the synergistic antibacterial effect of iron oxide nanoparticles and LNZ. OBJECTIVE: To study the release and antibacterial effects of LNZ-loaded superparamagnetic iron oxide nanoparticles (SPIONs) on Staphylococcus aureus and Streptococcus pneumoniae. METHOD: Ferrofluid containing SPIONs was synthesized via chemical co-precipitation method and stabilized by sodium lauryl sulphate (SLS). SPIONs were then loaded with LNZ and characterized for particle size, FT-IR, XRD, and entrapment efficiency. Further antibacterial activity of SPIONs and LNZ-loaded SPIONs was investigated. For the in vitro release findings, HPLC analytical method development and validation were performed. RESULTS: Isolation of LNZ was accomplished on a C-18 column with methanol-TBHS (tetra butyl ammonium hydrogen sulphate, 50:50, v/v). The eluate was monitored at 247 nm with a retention time of 4.175 min. The MNP's DLS measurement revealed monodispersed particles with an average size of 16.81 ± 1.07 nm and PDI 0.176 ± 0.012. In optimized formulation, 25 ± 1.75% (w/w) of the drug was found to be entrapped. XRD revealed uniform coating of oleic acid covering the entire magnetic particles' surface with no change in its crystallinity. An effective antimicrobial activity was observed at the lowered dose of drug. CONCLUSIONS: A robust HPLC method was developed to quantify the LNZ in MNPs, and outcomes showed that the reduced dose of LNZ incorporated in SPIONs was able to show similar activity as the marketed product. HIGHLIGHTS: Successfully reduction of the dose of LNZ was established with the aid of biocompatible MNPs to attain the equivalent antibacterial activity.