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Summary: The generation of knockins is fundamental to dissect biological systems. SEED/Harvest, a technology based on CRISPR-Cas9, offers a powerful approach for seamless genome editing in Drosophila. Here, we present a protocol to tag any gene in the Drosophila genome using SEED/Harvest technology. We describe knockin design, plasmid preparation, injection, and insertion screening. We then detail procedures for germline harvesting. The technique combines straightforward cloning and robust screening of insertions, while still resulting in scarless gene editing.For complete details on the use and execution of this protocol, please refer to Aguilar et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Although well-known for their thromboresistance, bileaflet mechanical heart valves (BMHV) require lifelong anti-thrombotic therapy. This must be associated with a certain level of thrombogenicity. Since both thromboresistance and thrombogenicity are explained by the blood-artificial surface or liquid-solid interactions, the aim of the present study was to explore BMHV thromboresistance from new perspectives. The wettability of BMHV pyrolytic carbon (PyC) occluders was investigated in under-liquid conditions. The submerged BMHV wettability clarifies the mechanisms involved in the thromboresistance. Methods The PyC occluders of a SJM Regent™ BMHV were previously laser irradiated, to create a surface hierarchical nano-texture, featuring three nano-configurations. Additionally, four PyC occluders of standard BMHV (Carbomedics, SJM Regent™, Bicarbon™, On-X®), were investigated. All occluders were evaluated in under-liquid configuration, with silicon oil used as the working droplet, while water, simulating blood, was used as the surrounding liquid. The under-liquid droplet-substrate wetting interactions were analyzed using contact angle goniometry. Results All the standard occluders showed very low contact angle, reflecting a pronounced affinity for non-polar molecules. No receding of the contact line could be observed for the untreated occluders. The smallest static contact angle of around 61° could be observed for On-X® valve (the only valve made of full PyC). The laser-treated occluders strongly repelled oil in underwater conditions. A drastic change in their wetting behaviour was observed depending on the surrounding fluid, displaying a hydrophobic behaviour in the presence of air (as the surrounding medium), and showing instead a hydrophilic nature, when surrounded by water. Conclusions BMHV “fear” water and blood. The intrinsic affinity of BMHV for nonpolar fluids can be translated into a tendency to repel polar fluids, such as water and blood. The blood-artificial surface interaction in BMHV is minimized. The contact between blood and BMHV surface is drastically reduced by polar-nonpolar Van der Waals forces. The “hydro/bloodphobia” of BMHV is intrinsically related to their chemical composition and their surface energy, thus their material: PyC indeed. Pertaining to thromboresistance, the surface roughness does not play a significant role. Instead, the thromboresistance of BMHV lies in molecular interactions. BMHV wettability can be tuned by altering the surface interface, by means of nanotechnology.

Background: Clavicle fractures are among the most common upper limb fractures in adults, with the midshaft region being the most frequently affected site. Minimally invasive plate osteosynthesis (MIPO) has emerged as an alternative to the traditional open reduction and internal fixation (ORIF) technique, offering potential advantages. The purpose of this study was to conduct a systematic review to explore the results of this technique in the existing literature, with emphasis on the occurrence of surgical complications and functional outcomes and also to provide a comprehensive comparison of MIPO and ORIF in the management of midshaft clavicle fractures. Methods: We conducted a systematic review to evaluate the complication incidence and clinical outcomes of MIPO for midshaft clavicle fractures. We searched PubMed/Medical Literature Analysis and Retrieval System Online (MEDLINE), Scopus, the Cochrane Database of Controlled Trials, and the Cochrane Database of Systematic Reviews databases without language or date restrictions. Studies focusing on midshaft clavicle fractures treated with MIPO were included, while other clavicle fractures and nonclinical studies were excluded. The risk of bias was assessed using the Methodological Index for Nonrandomized Studies criteria and the Risk of Bias Tool 2 Cochrane tool. Data synthesis included qualitative analysis, and if applicable, quantitative analysis and meta-analysis. Adherence to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines ensured reporting quality. Results: A total of 107 studies were initially identified, after applying inclusion and exclusion criteria, 22 studies were included for data extraction. These studies involved the evaluation of 714 clavicles treated with the MIPO technique. Of the 714 MIPO cases, 11 cases of implant failure, 5 nonunions, 2 infections, and 28 cases with neurological impairment were observed. Quantitative analysis comparing MIPO with ORIF revealed that MIPO had significantly shorter surgery time (mean difference −12.95, 95% confidence interval [−25.27 to −0.63], P = .04) and lower occurrence of numbness (odds ratio 0.29, 95% CI [0.15-0.56], P = .0002) compared to ORIF. Time to bone union, functional outcomes, and other complications were similar between MIPO and ORIF at the final follow-up. An overall moderate risk of bias was found across the studies. Conclusion: The MIPO technique yields good and comparable results to ORIF for midshaft clavicle fractures. Additionally, the MIPO technique may offer advantages such as reduced surgical time and lower chances of neurological impairment.

Purpose: Advancements in 3D printing technology have led to a growing interest on its application in orthopedic surgery. In the context of shoulder and elbow surgery, studies on 3D printing mostly center on surgical guides for the placement of the glenoid component in shoulder arthroplasty, but applications in non-arthroplasty procedures remain unclear. This systematic review aims to evaluate and summarize the literature on the current applications and clinical outcomes of 3D Patient-Specific Instruments (3DPSI) in non-arthroplasty procedures. We expected to find a predominant focus on corrective osteotomies with positive clinical outcomes and minimal complications. Methods: This systematic review adhered to PRISMA guidelines. Eligibility criteria included original research studies presenting primary data on 3DPSI for shoulder and elbow procedures. Exclusions were applied to studies exclusively reporting clinical data on 3DPSI for glenoid component placement in shoulder arthroplasty. A comprehensive literature search was conducted in PubMed/MEDLINE, Scopus, MedNar, Google Scholar, OAIster, and ProQuest Dissertations & Theses. Extracted data included study characteristics, 3DPSI development details, and clinical outcomes. Risk of bias was assessed using MINORS criteria. Results: Out of 845 initially identified records, the final analysis included 20 studies. Regarding the application of 3DPSI, 35 % of the reports addressed cubitus varus osteotomy correction, 30 % focused on clavicle malunion or nonunion, and 15 % centered on corrective osteotomies for proximal humerus malunion. Risk of bias assessment using MINORS criteria demonstrated a mean score of 9.11 out of 16 for studies without a comparator group. Results across different pathologies revealed high patient-reported outcomes (PROs), good patient satisfaction, and minimal complications, which are presented. Conclusion: In non-arthroplasty shoulder and elbow procedures, 3D Printed Patient-Specific Instrumentation have been mostly used for corrective osteotomies and demonstrates overall positive outcomes, low complications, and high patient satisfaction. Advancement in existing knowledge requires robust studies with larger cohorts and comparator groups. Level of Evidence: Level IV. Clinical Relevance: This study, summarizing existing data on 3D Patient-Specific Instruments (3DPSI) in non-arthroplasty shoulder and elbow procedures, offers guidance for future applications and research in this evolving field of orthopedic surgery.

Background: Phantom limb pain (PLP) is highly prevalent after amputation. However, the influence of non-painful sensations (PLS) remains unclear. This study examines the PLP-PLS index as a novel tool to differentiate PLP from PLS and explores the association of clinical factors with the index. Methods: We conducted a cross-sectional analysis of baseline data from 112 participants in a previous factorial trial in patients with unilateral traumatic lower limb amputation. Linear regression models were used to examine the associations between the index and various demographic, psychological and clinical factors. Logistic and Poisson regression, and e-value calculation were utilized for sensitivity analyses. Results: Adjusted multivariable linear regression models demonstrated significant associations of phantom movement sensation (β: −1.532; 95% CI: −2.615 to −0.449; p = 0.006) and time since amputation (β: 0.005; 95% CI: 0.0006 to 0.0101; p = 0.026) with the PLP-PLS index. These findings were confirmed by multivariable logistic regression (phantom movement sensation OR: 0.469; 95% CI: 0.200 to 1.099, p = 0.082; time since amputation OR: 1.003; 95% CI: 1.00003 to 1.007; p = 0.048) and sensitivity analyses. Conclusions: Time since amputation and phantom movement sensation likely reflect distinct phenotypes and potential mechanisms for PLP and PLS. The PLP-PLS index is a promising clinical tool for selecting therapies to prevent/treat PLP and for measuring treatment effects to modulate phantom pain. These findings emphasize the importance of understanding the mechanisms underlying PLP and PLS for improving clinical management and guiding future research.

Chemotherapy-induced peripheral neuropathy (CIPN) remains a clinical challenge for up to 80% of breast cancer survivors. In an open-label study, participants underwent three interventions: standard care (duloxetine) for 1 month (Phase 1), oral cannabidiol (CBD) for 2 months (Phase 2), and CBD plus multi-modal exercise (MME) for another 2 months (Phase 3). Clinical outcomes and gut microbiota composition were assessed at baseline and after each phase. We present the case of a 52-year-old female with a history of triple-negative breast cancer in remission for over five years presenting with CIPN. She showed decreased monocyte counts, c-reactive protein, and systemic inflammatory index after each phase. Duloxetine provided moderate benefits and intolerable side effects (hyperhidrosis). She experienced the best improvement and least side effects with the combined (CBD plus MME) phase. Noteworthy were clinically meaningful improvements in CIPN symptoms, quality of life (QoL), and perceived physical function, as well as improvements in pain, mobility, hand/finger dexterity, and upper and lower body strength. CBD and MME altered gut microbiota, showing enrichment of genera that produce short-chain fatty acids. CBD and MME may improve CIPN symptoms, QoL, and physical function through anti-inflammatory and neuroprotective effects in cancer survivors suffering from long-standing CIPN.

Summary: Introduction: rheumatoid arthritis (RA) is an autoimmune, inflammatory, and systemic disease, whose pathogenesis involves both the innate and adaptive immune systems. Its global prevalence varies from 0,4% to 1,3%, and the incidence increases with age. Given the increase in the general life expectancy of the population, the number of elderly patients with RA is growing, as are the complications of the disease. Several studies have demonstrated that patients with RA are at increased risk of developing lymphoproliferative diseases when compared to the general population. Some theories have emerged to explain this association, with emphasis on the role of the Epstein-Barr virus (EBV) and immunosuppressive drugs, especially methotrexate (MTX), the medication most frequently used to treat the disease. Methodology: Pubmed, Cochrane, Lilacs, Scopus, Embase and Web of Science databases were systematically reviewed in September 2023, without language or publication date restrictions. We searched for observational studies that associated EBV, RA, methotrexate and lymphoma. The recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the Cochrane Collaboration were used. Results: the analysis of 10 studies published between 2001 and 2022, involving 674 patients with RA and lymphoma, demonstrated that 77,3% used MTX and 27,4% were positive for EBV. The most common lymphoma was diffuse large B-cell lymphoma (DLBCL). The prevalence of EBV in DLBCL patients ranges from 5-15%, compared to 27,4% in the present study. This histological subtype was also associated with lower rates of spontaneous remission and worse outcomes. The prevalence of EBV in RA patients not treated with MTX was 7,7%, compared to 46% in patients who used the drug. Spontaneous remission rates (sRR) after MTX suspension (without associated immunochemotherapy) were variable (22,9% to 69,2%), but higher in the EBV-positive group. Discussion: in RA patients treated with MTX, EBV is expected to be activated through 3 mechanisms. The first of these involves AR itself. Patients with the disease have a deficiency of CD8+ T cells and, consequently, a high EBV burden due to immunodeficiency. The second mechanism is the suppression of T cell activation and adhesion by MTX. The third mechanism is direct reactivation of latent EBV by MTX. Thus, MTX withdrawal appears to result in recovery of immune surveillance and interruption of the EBV-induced tumor pathway. sRR with MTX withdraw were higher in EBV-positive group and could indicate that the virus is associated with a better outcome. The different prognosis of EBV-negative cases suggests that these tumors have a distinct biology. Conclusão: Conclusion: our systematic review found a high prevalence of EBV positivity in patients with RA and lymphoma, suggesting an important role for the virus in lymphomagenesis in this specific population. Due to the frequent use of MTX in the RA population, data from the systematic review alone are not capable of attributing the drug's causality to other confounding factors.

Abstract Advanced endometriosis is associated with a reduction of IVF success. Surgical damage to the ovarian reserve following the excision of endometriomas has been claimed as a critical factor in the explanation of this detrimental effect. However, it is generally inferred that other mechanisms might also hamper IVF success in affected women. They include diminished responsiveness to ovarian stimulation, altered steroidogenesis, a decline in oocyte quality, reduced fertilization and embryo development, and impaired implantation. To navigate these limitations, we scrutinized available literature for studies specifically designed to address distinct phases of the IVF process. Utmost consideration was given to intra-patient ovarian response comparisons in women with unilateral endometriomas and to studies applying a meticulous matching to control confounders. The following observations have been drawn: 1) endometriosis has a negligible impact on ovarian response. A slight reduction in stimulation response can only be observed for endometriomas larger than 4 cm. Follicular steroidogenesis is unaffected; 2) oocyte quality is not hampered. Fertilization rates are similar, and intracytoplasmic sperm injection (ICSI) is not justified. Embryonic development is uncompromised, with no increase in aneuploidy rate; 3) endometrial receptivity is either unaffected or only slightly impacted. In conclusion, our study suggests that, aside from the well-known negative effect on ovarian reserve from excisional endometrioma surgeries, endometriosis does not significantly affect IVF outcomes.