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Esophageal replacement (ER) in pediatric patients is complex, and while gastric or colonic interpositions are common, they carry long-term complications. Supercharged jejunal interposition (JI) offers better function but is technically challenging due to variable vasculature. We describe a protocol using computed tomography angiography (CTA) and 3D reconstruction to optimize surgical planning and vessel selection. A retrospective review of patients undergoing supercharged JI for ER was conducted. Preoperative CTA of the chest and neck assessed the internal thoracic arteries/veins (ITA/ITV) and secondary venous options, including cephalic (CV) and external jugular veins (EJV). CTA with 3D segmentation of the superior mesenteric vasculature mapped jejunal branching patterns. Thirteen patients underwent supercharged JI with CTA-based planning. The median age was 4.9 years (range: 1.5-15.5). Bilateral ITA visualization was achieved in all patients, and all underwent supercharge to the ITA. ITVs were visualized with lower resolution. Ten patients underwent venous anastomosis to the ITV. The CV was used three times due to insufficient ITV size. Of 26 potential vessels, 42% of CVs and 15% of EJVs were insufficient for use. Five patients underwent mesenteric imaging with 3D reconstruction which identified significant vascular variability. Preoperative vessel mapping correlated with intraoperative findings and reduced extensive mesenteric dissection. Preoperative CTA with 3D vascular modeling facilitates recipient and donor vessel selection, optimizes surgical planning, and may improve operative efficiency and outcomes in pediatric supercharged JI.

Supercharged proteins are some naturally occurring proteins that bear an abnormally high number of charged amino acids but can fold correctly and function normally. Owing to the nanoscale three-dimensional charge distribution associated with folding, the superpositively charged proteins possess the ability to rapidly enter mammalian cells with a potency much greater than cell-penetrating peptides and protein transduction domains. To harness the advantage, some proteins have been extensively mutated at their surface-exposed residues to generate "supercharged" variants such as +36GFP. Despite its effectiveness, this approach is time-consuming and only applicable to a few proteins that can tolerate the massive mutagenesis. Here, we proposed a tagging strategy to supercharge proteins and expand its application for the cytosolic delivery of proteins. Using the pan-protein binding ability of Coomassie brilliant blue (CBB), we developed a CBB-bearing cationic peptide CBB-R8 that can anchor on the protein surface and supercharge proteins without mutagenesis. The charge density of tagged proteins can be finely tuned by varying the tag/protein molar ratio, thus achieving a superior delivery efficiency to the cytosol than the canonical +36GFP. Mechanism studies revealed that the tagged protein could instantly and directly access the cytosol with little participation of endocytic pathways, resulting in the majority of delivered proteins being located in the cytosol. Moreover, the tagged proteins were discharged in the cytosol and return to biological functions after delivery, capable of catalyzing, blocking, and manipulating diverse targets inside cells. In conclusion, this simple plug-and-charge methodology greatly facilitates the preparation of supercharged proteins and expands its application to more intracellular targets.

This study presents a novel approach to enhance the sensitivity of a lateral flow immunoassay by computationally designing supercharged antibodies that optimize both the adsorption amount and molecular orientation on nanoparticle surfaces. We engineered immunoglobulin G antibodies with positively charged Fc domains and negatively charged Fab domains to create charge-polarized molecules for controlled interaction with negatively charged cellulose nanoparticles (NanoAct). The supercharged antibodies retained physicochemical properties and antigen-binding affinities identical to those of the wild-type antibody. Quantitative analysis showed that positively supercharged Fc domains enhanced antibody adsorption, and the charge-polarized design (featuring a negatively charged Fab and a positively charged Fc; c-10/Fc-pos14) enhanced relative Fab accessibility on the nanoparticle surface. Interaction analyses between supercharged antibodies and NanoAct using isothermal titration calorimetry quantitatively revealed that the c-10/Fc-pos14 antibody adsorbed onto NanoAct in the tail-on orientation. Consequently, lateral flow immunoassay performance tests demonstrated an 8-fold improvement in the limit of detection from 25 to 3.13 ng/mL, without increasing nonspecific binding. The key design principle involves maintaining sufficient charge separation between the domains to ensure proper orientation control. This supercharging approach represents a promising strategy for boosting immunoassay sensitivity while preserving sufficiently low noise levels with potential applications in other antibody-based diagnostic platforms.

Poly(ethylene terephthalate) (PET) is a highly recalcitrant polyester plastic whose resistance to degradation has contributed to widespread environmental accumulation. Enzymatic PET depolymerization has emerged as a promising bioremediation strategy, but PET hydrolysis remains challenging due to the insoluble and semi-crystalline nature of PET and the poor thermostability of many PET hydrolases at elevated temperatures. Here, several electrostatically supercharged PET binding modules (PBM) were fused to a PET-hydrolyzing Cutinase Catalytic Domain (CD) from the thermophilic microbe Thermobifida fusca to investigate how engineered PBM surface charge influences PET hydrolysis behavior. All PBM designs were derived from a native T. fusca family-2a carbohydrate binding module (CBM) as starting template. Since PET exhibited a substantially negative zeta potential, and accordingly, all positively supercharged PBMs displayed the strongest PET binding interactions in pull-down binding assays. However, stronger PET binding did not translate to improved hydrolysis activity for the fusion constructs. Instead, a slightly negatively charged PBM-CD fusion (D2 construct) exhibited activity comparable to the Cutinase CD on finely milled PET powder while showing substantially improved activity on intact PET discs, suggesting potential advantages for depolymerization of minimally processed PET feedstocks. Thermostability analysis identified an approximately 10 °C increase in melting temperature for the D2 fusion construct, corresponding to enhanced catalytic persistence and a shifted optimal hydrolysis temperature. Consequently, this construct exhibited an approximately 2-fold increase in long-term hydrolysis activity on milled PET and up to a 10-fold increase on intact PET discs, even at high solids loadings, compared to the native Cutinase CD. Collectively, these findings demonstrate that thermostability, rather than adsorption to PET alone, is a dominant factor governing functional persistence of PET hydrolases.

This study aims to compare demographic, risk, and complication profiles of pediatric and adult patients who underwent supercharged pedicled jejunal interposition for esophageal reconstruction.A systematic review and meta-analysis were performed, which included patients who underwent esophageal reconstruction with supercharged jejunum from 23 published studies. Patients were divided into two groups: pediatric/young adults (&#x2264;18 years), and adults (>18 years). The primary outcome was postoperative complications. Python 3.11 with pandas was used for data management, scikit-learn for Ridge regression and imputation of missing values, and SciPy for statistical analysis. Ridge regression analysis was utilized with regularization (&#x3b1;&#x2009;=&#x2009;0.1), while examining the relationship between demographic factors and overall complication rates in adult patients to account for limited sample sizes.A total of 254 manuscripts were reviewed, and 23 studies met inclusion criteria. Of 477 included patients, 415 were adults (87%) and 62 were pediatric patients (13%). Adult patients had significantly higher odds of developing an anastomotic leak (OR 8.63, p&#x2009;<&#x2009;0.01) and dysphagia (5.99, p&#x2009;<&#x2009;0.02) following surgery. Preoperative radiation was positively associated with postoperative dumping symptoms (&#x3b2;&#x2009;=&#x2009;0.56), stricture formation (&#x3b2;&#x2009;=&#x2009;0.27), poor wound healing (&#x3b2;&#x2009;=&#x2009;0.27), and need for reoperation (&#x3b2;&#x2009;=&#x2009;0.27). A history of cancer was most positively associated with anastomotic leak (&#x3b2;&#x2009;=&#x2009;0.22) following surgery. Preoperative radiation was positively associated with anastomotic leak (&#x3b2;&#x2009;=&#x2009;0.12). Smoking demonstrated a strong inverse association with the need for reoperation (&#x3b2;&#x2009;=&#x2009;-&#x2009;0.66), and a weaker inverse association with leakage (&#x3b2;&#x2009;=&#x2009;-&#x2009;0.25).Adult patients have a significantly greater likelihood of experiencing postoperative anastomotic leakage and dysphagia compared with pediatric patients. Preoperative radiation was associated with dumping symptoms, stricture, need for reoperation, poor wound healing, and pulmonary complications. Smoking was associated with decreased need for reoperation and anastomotic leakage.

Silica nanoparticles (SiNPs) produced during semiconductor manufacturing possess dimensions in the nanometer range with a high net negative surface charge, imparting them with exceptional colloidal stability. These, when released into the environment, persist for extended periods, leading to ecological and health risks. Although conventional treatment strategies such as coagulation-flocculation and membrane-based separations are effective, they depend on nonbenign chemical additives and are prone to fouling, respectively. Motivated by this, we introduce a bioadsorptive recovery strategy that exploits recombinantly expressed supercharged green fluorescent proteins (scGFPs), which act as an adsorbent to selectively capture SiNPs (adsorbate) through tunable electrostatic interactions under aqueous conditions. A panel of scGFP was systematically evaluated to validate electrostatics-driven adsorption onto silica nanoparticles. The highly charged protein variants act as multivalent linkers, simultaneously binding multiple silica nanoparticles and thereby inducing nanoparticle-nanoparticle bridging and aggregation. This was monitored through the intrinsic fluorescence of scGFP and a complementary colorimetric silicate ion assay. The critical role of electrostatics was highlighted through molecular dynamics and metadynamics simulations. The applicability of these proteins was demonstrated through single-batch adsorption with a reuse capacity of up to three times. Additionally, to translate this interaction into a solid-phase recovery system, silica microparticles were sequentially coated with scGFP and assessed for their ability to adsorb SiNPs in a simple single-pass column adsorption. Together, these findings establish a tunable, charge-driven, and fully aqueous protein-based platform for SiNP recovery, offering a promising alternative to conventional separation methods used in semiconductor wastewater treatment.

Cell-penetrating peptides (CPPs) and supercharged proteins (SPs) enable efficient intracellular delivery of macromolecules, with expanding applications in basic research and in therapeutic development. Despite their potential, reproducible workflows for isolation, biochemical characterization, and quantitative uptake analysis remain limited. Here, we present a comprehensive and replicable protocol for the isolation, characterization, and cellular uptake analysis of CPP-fusion proteins (CPP-FPs) and SPs using methyl-CpG-binding protein 2 (MeCP2) constructs as a proof-of-principle model. This workflow combines native protein purification with dynamic light scattering (DLS)-based buffer optimization. Cellular uptake is then assessed and quantified under live-cell conditions using high-content imaging and imaging flow cytometry, with additional assays to probe endocytic trafficking routes, identify CPP-like motifs in SPs, and validate transducing CPP-FP/SP functionality. The protein isolation and DLS-guided buffer screen yield samples with long-term stability. Live-cell fluorescence microscopy and imaging flow cytometry enable discrimination between membrane-bound and internalized signal, providing higher accuracy compared to plate-based readouts. MeCP2 sequence probing has revealed the presence of a CPP-like motif that is critical to its internalization. Finally, validation assays clearly demonstrated CPP-FP/SP activity. This protocol integrates advances in protein biochemistry, structural analysis, and live-cell imaging into a reproducible pipeline adaptable to a wide range of CPP- and SP-based protein constructs and provides a practical framework for downstream mechanistic and therapeutic interventions.

High-definition (HD) liposuction is widely favored by plastic surgeons and patients for its capacity to create athletic, sculpted body contours. However, traditional HD liposuction may not effectively enhance muscle volume, resulting in a less natural appearance during motion. To evaluate the benefits and efficacy of the Supercharged Body Contouring (S-Bc) technique, which employs endoscopic intramuscular fat grafting to augment the rectus abdominis and external oblique muscles under direct endoscopic visualization. From October 2019 and November 2024, 32 patients (30 male, 2 female) underwent abdominal deep plane liposuction with preservation of superficial fat, combined with the S-Bc technique, a novel endoscopic intramuscular fat grafting method designed to enhance abdominal muscle definition. No significant complications, such as infections, pulmonary embolism, venous embolism, or fat embolism, were reported. A minor complication, mild seroma, occurred in 8 patients. Other minor issues included temporary bruising in 2 patients and slight asymmetry in one, which was addressed with a secondary fat injection at 3&#x2005;months. At the 1-year follow-up, the median patient satisfaction score was 9.5 out of 10 (range: 7-10, 95% CI: 9-10). The fat injection technique for enhancing muscle appearance produced sustained results in all patients. Skin tightening evaluations showed 56.25% (n = 18) of patients rated as good, 40.6% (n = 13) as very good, and 3.12% (n = 1) as moderate. The S-Bc technique offers a safe and reproducible method for enhancing HD liposuction, achieving natural and dynamic abdominal contours. This case series suggests its applicability in aesthetic abdominal surgery, pending further validation in larger studies.

Supercharge end-to-side (SETS) nerve transfer enhances motor recovery in proximal nerve injuries by providing early reinnervation. However, the optimal indications and mechanisms remain unclear. This study examined the role of donor nerves using rat models of varying injury severity to clarify the clinical indications for SETS. Eighty female Sprague-Dawley rats were assigned to five groups: Control, Mild-SETS(-), Mild-SETS(+), Severe-SETS(-), and Severe-SETS(+). The tibial nerve was transected, decellularized, and reconstructed with a 10 mm (mild) or 20 mm (severe) graft. SETS consisted of end-to-side coaptation of the donor peroneal nerve to the tibial nerve 5 mm distal to the graft. Assessments included the sciatic functional index (SFI; measured every 4 weeks), compound muscle action potentials (CMAPs), gastrocnemius weight, and immunostaining for neurofilament (NF)-positive axons and S100&#x3b2;-positive Schwann cells at 8 and 16 weeks. In mild models, SETS accelerated early recovery in CMAP amplitude and muscle weight without affecting long-term outcomes. In severe models, SETS showed significant increases in CMAP amplitude and muscle weight at 16 weeks. NF-positive axons and S100&#x3b2;-positive Schwann cells increased distal to the coaptation site at 8 and 16 weeks in mild models, whereas both distal and proximal increases were observed in severe models. Donor nerves in SETS enable early arrival of axons and Schwann cells, leading to faster motor improvement. In the long term, spontaneous recovery compensates in mild models, whereas severe models benefit from sustained donor support that promotes regeneration. SETS nerve transfer may therefore be particularly useful in selected mild cases where rapid recovery is desired, and especially in severe cases where spontaneous regeneration is insufficient.

The Outerbridge-Kashiwagi (O-K) procedure has been widely used to treat elbow osteoarthritis by improving range of motion and alleviating mechanical impingement. Supercharged end-to-side (SETS) anterior interosseous nerve (AIN) transfer has emerged as a promising technique for restoring hand muscle function in patients with ulnar nerve palsy. However, no previous studies have evaluated the combined use of these procedures in patients with both elbow arthritis and severe ulnar neuropathy presenting with intrinsic hand muscle atrophy. We hypothesized that the combination of the O-K procedure and SETS AIN transfer would improve both elbow mobility and hand motor strength. A retrospective analysis was performed on 22 patients treated between 2019 and 2023 who underwent a miniopen O-K procedure, cubital tunnel release with anterior transposition, and SETS AIN-to-ulnar motor branch transfer. Inclusion criteria included McGowan grade 3 cubital tunnel syndrome, limited elbow range of motion (ROM) below functional thresholds, and ulnar-innervated intrinsic muscle weakness (MRC grade 0-3) with axonal loss on electromyography. Functional outcomes were evaluated using elbow ROM, MRC grading of the first dorsal interosseous (FDI), grip and pinch strength, index and little finger abduction/adduction strength, and Disabilities of the Arm, Shoulder, and Hand (DASH) scores. At final follow-up, significant improvements were observed in elbow ROM (from 80.9&#xb0; to 106.5&#xb0;, P < 0.001), FDI-MRC grade (2.32-3.23, P < 0.001), grip and pinch strength, and DASH scores (P < 0.001). Notably, greater improvement in DASH scores was seen in cases involving the dominant hand. Radiographic assessment revealed sustained fenestration, and no surgical complications were reported. This combined approach addresses both mechanical impingement and motor deficits, offering a feasible and effective strategy for restoring upper extremity function in this patient population. Preoperative electrophysiologic assessment plays a key role in optimizing candidate selection and outcomes.

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Targeted protein degradation modulates protein function beyond the inhibition of enzyme activity or protein-protein interactions. Most degrader drugs function by directly mediating the proximity between a neosubstrate and a hijacked E3 ligase. Here we identify pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce the formation of kynurenine by both inhibition and induced degradation of the enzyme and thus also modulate the non-enzymatic functions of IDO1. This unique mechanism of action may open up alternative therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

Biofilms formed by flora can be sustainably applied in a variety of fields such as bioremediation, wastewater treatment, corrosion prevention, and agricultural production. However, highly practical biofilms often result in low microbial activity, due to undesired impacts including environmental stress and microbial competition. Leveraging the advantages of carrier materials, we aimed to enhance the degradation efficiency and resilience of biofilms by integrating biochar. In this study, the biochar with excellent economic benefits and adsorption capacity was prepared and selected as the carrier material. The growth characteristics, pollutant removal performance, and nutrient cycling within biochar-based biofilms were systematically investigated. The result validated the pollutant remediation efficiency of biofilms increased by 14&#x223c;18&#x202f;% after adding biochar, and found a positive nutrient cycling existing within the biochar-based biofilms. Subsequently, the enhanced remediation mechanisms of biochar-based biofilms at the molecular level were explored through metagenomic and metabolomic analyses. Our results indicate superior strengths of biochar-based biofilms in both metabolic activity and beneficial genes compared to monocultured biofilms. This study aims to improve the stability of biofilms formed by functional flora and reveal their potential in bioremediation for contaminants.

Sweeping study finds rapid genetic change in past 10,000 years.

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Light-driven molecular motors undergo directional motions upon the input of external energy and represent archetypical molecular machines. So far, such motors have functioned via light-induced bond rotations, where directionality is dictated by a fixed source of asymmetry. During the operation cycle, no further structural changes occur, other than the rotation itself. Here we disclose a highly effective mechanism for light-driven motor rotation involving constitutional alteration and reversible proton transfer. Associated with this unusual mechanism is a particularly high energy content from the incident light that the motor retains. This feature is further exploited in a low-temperature molecular solar thermal energy storage application, where solar energy can be stored and released in a controlled fashion and tracked step by step with the naked eye. With these findings, unique possibilities emerge for the design and use of molecular motors with hitherto unknown modes of action and power.

Natural killer (NK) cells are pivotal effectors in innate anti-tumor immunity, but their efficacy against solid tumors is constrained by inadequate tumor infiltration and functional suppression within the tumor microenvironment (TME). Although ex vivo expansion increases NK cell numbers, poor tumor homing and transient post-infusion activity persist as major limitations. This work aims to develop a combinatorial approach integrating ex vivo NK cell expansion with localized immunomodulation via engineered oncolytic adenoviruses (oAds) to address these challenges. K562 feeder cells were engineered to stably express IL-2, membrane-bound IL-21 (mbIL-21), and 4-1BBL to activate and expand NK cells ex vivo. Following irradiation, these cells were used to expand NK cells ex vivo. Armed oAds (oAd-IL-2/mbIL-21/4-1BBL) were designed to express IL-2, mbIL-21, and 4-1BBL. In vitro assays were used to evaluate the impact of oAd-IL-2/mbIL-21/4-1BBL on tumor cell lysis, as well as NK cell proliferation, activation, and migration. A HCT116 subcutaneous tumor-bearing mouse model was used to assess the combined anti-tumor effects of ex vivo-expanded NK cells and oAd-IL-2/mbIL-21/4-1BBL, focusing on tumor growth inhibition and NK cell infiltration in tumor lesions. We firstly constructed K562 feeder cells stably co-express IL-2, mbIL-21, and 4-1BBL, enabling 100-fold NK cell expansion (>85% purity) within 14&#xa0;days. Concurrently, armed oAds were engineered to deliver these immunomodulators. oAd-IL-2/mbIL-21/4-1BBL enhanced NK cell proliferation, activation, migration, and tumor cell lysis in vitro. In HCT116 colorectal xenograft models, combing ex vivo-expanded NK cells with oAd-IL-2/mbIL-21/4-1BBL synergistically suppressed tumor growth and increased tumor-infiltrating NK cells. Mechanistically, oAds elicited a "self-feeder" effect through localized immunomodulator production, sustaining NK cell activity within the TME. These findings define a dual-phase strategy that integrate scalable ex vivo expansion with in situ activation to overcome key barriers in NK cell therapy for solid tumors.

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Health insurers and health care provider organizations are increasingly using artificial intelligence (AI) tools in prior authorization and claims processes. AI offers many potential benefits, but its adoption has raised concerns about the role of the "humans in the loop," users' understanding of AI, opacity of algorithmic determinations, underperformance in certain tasks, automation bias, and unintended social consequences. To date, institutional governance by insurers and providers has not fully met the challenge of ensuring responsible use. However, several steps could be taken to help realize the benefits of AI use while minimizing risks. Drawing on empirical work on AI use and our own ethical assessments of provider-facing tools as part of the AI governance process at Stanford Health Care, we examine why utilization review has attracted so much AI innovation and why it is challenging to ensure responsible use of AI. We conclude with several steps that could be taken to help realize the benefits of AI use while minimizing risks.