Development of a zoledronate-modified functional alginate hydrogel with effects of promoting osteogenesis for potential osteoporosis treatment.

Osteoporosis is one of the most prevalent age-related diseases worldwide. It is characterized by a systemic deterioration in bone strength (bone density and bone mass), leading to an increase in fragility fractures. The complex pathological environment of osteoporosis presents a significant challenge to the induction of bone regeneration under osteoporotic conditions. Therefore, the development of a system for local delivery of active substances with osteoinductive effects is of practical significance in the clinical treatment of osteoporosis. In this study, we successfully loaded the anti-osteoporotic small molecule drug zoledronate (ZOL) into calcium alginate to prepare a biologically functional hydrogel, designated as ALG-ZOL-Ca. The prepared ALG-ZOL-Ca hydrogel gels quickly, making the hydrogel easy to inject and adapt to irregularly shaped bone defects, and simultaneously exhibits good bioactivity and osteoconductivity. The RT-qPCR results suggested that this hydrogel effectively promoted the expression levels of ß-catenin and Axin2, which indicating a stimulative effect on the Wnt/ß-catenin pathway in vitro. Moreover, ALG-ZOL-Ca hydrogel effectively promoted the expression of the OCN and SP7 genes. Therefore, this study proposes a new functional composite hydrogel that provides a potential treatment strategy for osteoporosis.

Efficacy of Foldable Capsular Vitreous Body Implants Filled With Light or Heavy Silicone Oil in the Treatment of Silicone Oil-dependent Eyes.

Purpose: The purpose of this study was to compare the clinical efficacy of foldable capsular vitreous body (FCVB) filled with either light or heavy silicone oil and the incidence of complications after their implantation for the treatment of severe ocular trauma and silicone oil-dependent eyes. Methods: FCVB filled with either light (n = 16) or heavy (n = 8) silicone oil was implanted in 24 patients. During the 12-month follow-up period, the intraocular pressure, final best-corrected visual acuity, retinal reattachment condition, position of the FCVB, and complications were assessed. Results: All surgeries were performed without issue. There was no significant difference in preoperative and postoperative best-corrected visual acuity between the two groups. A significant improvement in the intraocular pressure was observed after surgery in both the light silicone oil (P = 0.029) and heavy silicone oil (P = 0.035) groups. None of the patients developed displacement or prolapse of the FCVB. The most common early and late postoperative complications were postoperative hemorrhage (33.3%) and corneal opacification (50%), respectively. Conclusions: FCVB filled with heavy silicone oil can be used as a supplemental therapy for patients who have lost the anterior segment of their eye, have lesions of the inferior retina, or cannot maintain the prone position for various reasons. Translational Relevance: Implantation of FCVB combined with heavy silicone oil compensates for the shortcomings of this with light silicone oil, providing patients with more personalized treatment.

Association of lifestyle with valvular heart disease progression and life expectancy among elderly people from different socioeconomic backgrounds.

BACKGROUND: Current cardiovascular prevention strategies are based on studies that seldom include valvular heart disease (VHD). The role of modifiable lifestyle factors on VHD progression and life expectancy among the elderly with different socioeconomic statuses (SES) remains unknown. METHODS: This cohort study included 164,775 UK Biobank participants aged 60 years and older. Lifestyle was determined using a five-factor scoring system covering smoking status, obesity, physical activity, diet, and sleep patterns. Based on this score, participants were then classified into "poor," "moderate," or "ideal" lifestyle groups. SES was classified as high or low based on the Townsend Deprivation Index. The association of lifestyle with major VHD progression was evaluated using a multistate mode. The life table method was employed to determine life expectancy with VHD and without VHD. RESULTS: The UK Biobank documented 5132 incident VHD cases with a mean follow-up of 12.3 years and 1418 deaths following VHD with a mean follow-up of 6.0 years. Compared to those with a poor lifestyle, women and men followed an ideal lifestyle had lower hazard ratios for incident VHD (0.66 with 95% CI, 0.59-0.73 for women and 0.77 with 95% CI, 0.71-0.83 for men) and for post-VHD mortality (0.58 for women, 95% CI 0.46-0.74 and 0.62 for men, 95% CI 0.54-0.73). When lifestyle and SES were combined, the lower risk of incident VHD and mortality were observed among participants with an ideal lifestyle and high SES compared to participants with an unhealthy lifestyle and low SES. There was no significant interaction between lifestyle and SES in their correlation with the incidence and subsequent mortality of VHD. Among low SES populations, 60-year-old women and men with VHD who followed ideal lifestyles lived 4.2 years (95% CI, 3.8-4.7) and 5.1 years (95% CI, 4.5-5.6) longer, respectively, compared to those with poor lifestyles. In contrast, the life expectancy gain for those without VHD was 4.4 years (95% CI, 4.0-4.8) for women and 5.3 years (95% CI, 4.8-5.7) for men when adhering to an ideal lifestyle versus a poor one. CONCLUSIONS: Adopting a healthier lifestyle can significantly slow down the progression from free of VHD to incident VHD and further to death and increase life expectancy for both individuals with and without VHD within diverse socioeconomic elderly populations.

A pan-allelic human SIRPα-blocking antibody, ES004-B5, promotes tumor killing by enhancing macrophage phagocytosis and subsequently inducing an effective T-cell response.

As a major immune cell type in the tumor microenvironment, tumor-associated macrophages secrete suppressive factors that can inhibit antitumor immunity and promote tumor progression. One approach trying to utilize macrophages for immunotherapy has been to block the CD47-SIRPα axis, which mediates inhibitory signaling, to promote phagocytosis of tumor cells. Many CD47-targeted agents, namely, anti-CD47 antibodies and SIRPα fusion proteins, were associated with a diverse spectrum of toxicities that limit their use in clinical settings. Universal expression of CD47 also leads to a severe "antigen sink" effect of CD47-targeted agents. Given that the CD47 receptor, SIRPα, has a more restricted expression profile and may have CD47-independent functions, targeting SIRPα is considered to have distinct advantages in improving clinical efficacy with a better safety profile. We have developed ES004-B5, a potentially best-in-class pan-allelic human SIRPα-blocking antibody using hybridoma technology. ES004-B5 binds to major human SIRPα variants through a unique epitope with high affinity. By blocking CD47-induced inhibitory "don't-eat-me" signaling, ES004-B5 exerts superior antitumor activity in combination with anti-tumor-associated antigen antibodies in vitro and in vivo. Unlike CD47-targeted agents, ES004-B5 exhibits an excellent safety profile in nonhuman primates. ES004-B5 has potential to be an important backbone for SIRPα-based combination therapy and/or bispecific antibodies, which will likely overcome the limitations of CD47-targeted agents encountered in clinical settings.

Innovative Application of the Ilizarov Technique for Long Nerve Defect Reconstruction: A Detailed Case Report.

BACKGROUND: Traditionally known for bone regeneration, the Ilizarov technique's effectiveness in nerve reconstruction, particularly for extensive nerve damage, has yet to be widely recognized. CASE PRESENTATION: This report presents a case study and proposes the innovative use of the Ilizarov technique for reconstructing extended nerve defects. In this study, we reviewed a 43-year-old male diagnosed with an open fracture of the right tibia combined with soft tissue injury resulting in a mangled injury in which a large part of his right tibial bone and nerve were lost. The patient was cured and the sensorimotor function was recovered after distraction osteogenesis by the Ilizarov technique, which is a unique application of this technique to repair a substantial long nerve defect, a rare occurrence in medical literature. It highlights the method of nerve lengthening, which is achieved by attaching the nerve stump to the bone stump. This approach allows for significant nerve regeneration and ensures a stable progression of the nerve, as the bone stump acts as a carrier, overcoming the challenges of direct nerve lengthening. CONCLUSIONS: The adaptability and effectiveness of the Ilizarov technique in a new area suggests the need to reconsider traditional approaches to complex nerve reconstruction. Placing this case within the context of current medical knowledge underscores the potential of this technique to revolutionize the treatment of extended nerve defects, offering hope for improved outcomes in challenging scenarios.

Sequential delivery of photosensitizers and checkpoint inhibitors by engineered bacteria for enhanced cancer photodynamic immunotherapy.

Engineered bacteria-based cancer therapy has increasingly been considered to be a promising therapeutic strategy due to the development of synthetic biology. Wherein, engineering bacteria-mediated photodynamic therapy (PDT)-immunotherapy shows greater advantages and potential in treatment efficiency than monotherapy. However, the unsustainable regeneration of photosensitizers (PSs) and weak immune responses limit the therapeutic efficiency. Herein, we developed an engineered bacteria-based delivery system for sequential delivery of PSs and checkpoint inhibitors in cancer PDT-immunotherapy. The biosynthetic pathway of 5-aminolevulinic acid (5-ALA) was introduced into Escherichia coli, yielding a supernatant concentration of 172.19 mg/L after 10 h of growth. And another strain was endowed with the light-controllable releasement of anti-programmed cell death-ligand 1 nanobodies (anti-PD-L1). This system exhibited a collaborative effect, where PDT initiated tumor cell death and the released tumor cell fragments stimulated immunity, followed by the elimination of residual tumor cells. The tumor inhibition rate reached 74.97%, and the portion of activated T cells and inflammatory cytokines were reinforced. The results demonstrated that the engineered bacteria-based collaborative system could sequentially deliver therapeutic substance and checkpoint inhibitors, and achieve good therapeutic therapy. This paper will provide a new perspective for the cancer PDT-immunotherapy.

The STAT1-SLC31A1 axis: Potential regulation of cuproptosis in diabetic retinopathy.

BACKGROUND: By identifying molecular biological markers linked to cuproptosis in diabetic retinopathy (DR), new pathobiological pathways and more accessible diagnostic markers can be developed. METHODS: The datasets related to DR were acquired from the Gene Expression Omnibus database, while genes associated with cuproptosis were sourced from previously published compilations. Consensus clustering was conducted to delineate distinct DR subclasses. Feature genes were identified utilizing weighted correlation network analysis (WGCNA). Additionally, two machine-learning algorithms were employed to refine the selection of feature genes. Finally, we conducted preliminary validation experiments to ascertain the involvement of cuproptosis in DR development and the transcriptional regulation of critical genes using both the streptozotocin-induced diabetic mouse model and the high glucose-induced BV2 model. RESULTS: In the STZ-induced diabetic mouse retinas, a decrease in the expression of cuproptosis signature proteins (FDX1, DLAT, and NDUFS8) suggested the occurrence of cuproptosis in DR. Subsequently, the expression of eight cuproptosis differential genes was validated through the STZ-induced diabetes and oxygen-induced retinopathy (OIR) models, with the key gene SLC31A1 showing upregulation in both models and dataset species. Further analyses, including weighted gene co-expression network analysis, GSVA, and immune infiltration analysis, indicated a close correlation between cuproptosis and microglia function. Additionally, validation in an in vitro model of microglia indicated the occurrence of cuproptosis in microglia under high glucose conditions, alongside abnormal expression of STAT1 with SLC31A1. CONCLUSION: Our findings suggest that STAT1/SLC31A1 may pave the way for a deeper comprehension of the mechanistic basis of DR and offer potential therapeutic avenues.

Exploring potential predictive biomarkers through historical perspectives on the evolution of systemic therapies into the emergence of neoadjuvant therapy for the treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a type of liver cancer, ranks as the sixth most prevalent cancer globally and represents the third leading cause of cancer-related deaths. Approximately half of HCC patients miss the opportunity for curative treatment and are then limited to undergoing systemic therapies. Currently, systemic therapy has entered the era of immunotherapy, particularly with the advent of immune-checkpoint inhibitors (ICIs), which have significantly enhanced outcomes for patients with advanced HCC. Neoadjuvant treatment for HCC has become a possibility-findings from the IMbrave 050 trial indicated that ICIs offer the benefit of recurrence-free survival for high-risk HCC patients post-resection or local ablation. However, only a small fraction of individuals benefit from systemic therapy. Consequently, there is an urgent need to identify predictive biomarkers for treatment response and outcome assessment. This study reviewed the historical progression of systemic therapy for HCC, highlighting notable therapeutic advancements. This study examined the development of systemic therapies involving conventional drugs and clinical trials utilized in HCC treatment, as well as potential predictive biomarkers for advanced and/or locally advanced HCC. Various studies have revealed potential biomarkers in the context of HCC treatment. These include the association of dendritic cells (DCs) with a favorable response to neoadjuvant therapy, the presence of enriched T effector cells and tertiary lymphoid structures, the identification of CD138+ plasma cells, and distinct spatial arrangements of B cells in close proximity to T cells among responders with locally advanced HCC receiving neoadjuvant cabozantinib and nivolumab treatment. Furthermore, pathological response has been associated with intratumoral cellular triads consisting of progenitor CD8+ T cells and CXCL13+ CD4+ T helper cells surrounding mature DCs in patients receiving neoadjuvant cemiplimab for resectable HCC. Despite no widely recognized predictive biomarkers for HCC individualized treatment, we believe neoadjuvant trials hold the most promise in identifying and validating them. This is because they can collect multiple samples from resectable HCC patients across stages, especially with multi-omics, bridging preclinical and clinical gaps.

Therapeutic potential of flavopiridol in diabetic retinopathy: Targeting DDX58.

Diabetic retinopathy (DR), a common complication of diabetes, is characterized by inflammation and neovascularization, and is intricately regulated by the ubiquitin-proteasome system (UPS). Despite advancements, identifying ubiquitin-related genes and drugs specifically targeting DR remains a significant challenge. In this study, bioinformatics analyses and the Connectivity Map (CMAP) database were utilized to explore the therapeutic potential of genes and drugs for DR. Through these methodologies, flavopiridol was identified as a promising therapeutic candidate. To evaluate flavopiridol's therapeutic potential in DR, an in vitro model using Human Umbilical Vein Endothelial Cells (HUVECs) induced by high glucose (HG) conditions was established. Additionally, in vivo models using mice with streptozotocin (STZ)-induced DR and oxygen-induced retinopathy (OIR) were employed. The current study reveals that flavopiridol possesses robust anti-inflammatory and anti-neovascularization properties. To further elucidate the molecular mechanisms of flavopiridol, experimental validation and molecular docking techniques were employed. These efforts identified DDX58 as a predictive target for flavopiridol. Notably, our research demonstrated that flavopiridol modulates the DDX58/NLRP3 signaling pathway, thereby exerting its therapeutic effects in suppressing inflammation and neovascularization in DR. This study unveils groundbreaking therapeutic agents and innovative targets for DR, and establishes a progressive theoretical framework for the application of ubiquitin-related therapies in DR.

Surface bioactivation of Polyetheretherketone (PEEK) by magnesium chondroitin sulfate (MgCS) as orthopedic implants for reconstruction of skeletal defects.

Polyether-ether-ketone (PEEK) is clinically used as a bio-implant for the healing of skeletal defects. However, the osseointegration of clinical-sized bone grafts remains limited. In this study, surface-porous PEEK was created by using a sulfonation method and a metal-polysaccharide complex MgCS was introduced on the surface of sulfonated PEEK to form MgCS@SPEEK. The as-prepared MgCS@SPEEK was found to have a porous surface with good hydrophilicity and bioactivity. This was followed by an investigation into whether MgCS loaded onto sulfonated PEEK surfaces could promote osseointegration and angiogenesis. The in vitro results showed that MgCS@SPEEK had a positive effect on reducing the expression levels of inflammatory genes and promoting osteogenesis and angiogenesis-related genes expression levels. Furthermore, porous MgCS@SPEEK was implanted in critical-sized rat tibial defects for in vivo evaluation of osseointegration. The micro-computed tomography evaluation results revealed substantial bone formation at 4 and 8 weeks. Collectively, these findings indicate that MgCS@SPEEK could provide improved osseointegration and an attractive strategy for orthopaedic applications.

Chronic Postsurgical Pain Following Lung Transplantation: Characteristics, Risk Factors, Treatment, and Prevention: A Narrative Review.

Chronic pain after lung transplantation (LTx) can substantially reduce quality of life (QoL), yet current consensus guidelines say little about how to prevent or manage it. Research on pain after LTx has tended to focus on acute rather than chronic pain, and it has not extensively examined the factors associated with onset or resolution of chronic pain, which differ from factors influencing chronic pain after general thoracic surgery. This narrative review explores what is known about the epidemiology and risk factors of chronic pain after LTx, as well as effective ways to treat or prevent it. The review identifies key questions and issues that should be the focus of future research.

Predictive modeling for identifying infection risk following spinal surgery: Optimizing patient management.

Infection is known to occur in a substantial proportion of patients following spinal surgery and predictive modeling may provide a useful means for identifying those at higher risk of complications and poor prognosis, which could help optimize pre- and postoperative management strategies. The outcome measure of the present study was to investigate the occurrence of all-cause infection during hospitalization following scoliosis surgery. To meet this aim, the present study retrospectively analyzed 370 patients who underwent surgery at the Second Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) between January 2016 and October 2022, and patients who either experienced or did not experience all-cause infection while in hospital were compared in terms of their clinicodemographic characteristics, surgical variables and laboratory test results. Logistic regression was subsequently applied to data from a subset of patients in order to build a model to predict infection, which was validated using another subset of patients. All-cause, in-hospital postoperative infections were found to have occurred in 66/370 patients (17.8%). The following variables were included in a predictive model: Sex, American Society of Anesthesiologists (ASA) classification, body mass index (BMI), diabetes mellitus, hypertension, preoperative levels of white blood cells and preoperative C-reactive protein (CRP) and duration of surgery. The model exhibited an area under the curve of 0.776 against the internal validation set. In conclusion, dynamic nomograms based on sex, ASA classification, BMI, diabetes mellitus, hypertension, preoperative levels of white blood cells and CRP and duration of surgery may have the potential to be a clinically useful predictor of all-cause infection following scoliosis. The predictive model constructed in the present study may potentially facilitate the real-time visualization of risk factors associated with all-cause infection following surgical procedures.

Bioinformatics analysis for constructing a cellular senescence-related age-related macular degeneration diagnostic model and identifying relevant disease subtypes to guide treatment.

Age-related macular degeneration (AMD) is a condition causing progressive central vision loss. Growing evidence suggests a link between cellular senescence and AMD. However, the exact mechanism by which cellular senescence leads to AMD remains unclear. Employing machine learning, we established an AMD diagnostic model. Through unsupervised clustering, two distinct AMD subtypes were identified. GO, KEGG, and GSVA analyses explored the diverse biological functions associated with the two subtypes. By WGCNA, we constructed a coexpression network of differential genes between the subtypes, revealing the regulatory role of hub genes at the level of transcription factors and miRNAs. We identified 5 genes associated with inflammation for the construction of the AMD diagnostic model. Additionally, we observed that the level of cellular senescence and pathways related to programmed cell death (PCD), such as ferroptosis, necroptosis, and pyroptosis, exhibited higher expression levels in subtype B than A. Immune microenvironments also differed between the subtypes, indicating potentially distinct pathogenic mechanisms and therapeutic targets. In summary, by leveraging cellular senescence-associated gene expression, we developed an AMD diagnostic model. Furthermore, we identified two subtypes with varying expression patterns of senescence genes, revealing their differential roles in programmed cell death, disease progression, and immune microenvironments within AMD.

A New Modulator of Neuroinflammation in Diabetic Retinopathy: USP25.

Diabetic retinopathy (DR) is a diabetes-associated complication that poses a threat to vision, distinguished by persistent and mild inflammation of the retinal microvasculature. The activation of microglia plays a crucial role in driving this pathological progression. Previous investigations have demonstrated that ubiquitin-specific peptidase 25 (USP25), a deubiquitinating enzyme, is involved in the regulation of immune cell activity. Nevertheless, the precise mechanisms through which USP25 contributes to the development of DR remain incompletely elucidated. Firstly, we have demonstrated the potential mechanism by which ROCKs can facilitate microglial activation and augment the synthesis of inflammatory mediators through the modulation of NF-κB signaling pathways in a high-glucose milieu. Furthermore, our study has provided novel insights by demonstrating that the regulatory role of USP25 in the secretion of proinflammatory factors is mediated through the involvement of ROCK in modulating the expression of NF-κB and facilitating the nuclear translocation of the phosphatase NF-κB. This regulatory mechanism plays a crucial role in modulating the activation of microglial cells within a high-glycemic environment. Hence, USP25 emerges as a pivotal determinant for the inflammatory activation of microglial cells, and its inhibition exhibits a dual effect of promoting retinal neuron survival while suppressing the inflammatory response in the retina. In conclusion, the promotion of diabetic retinopathy (DR) progression by USP25 is attributed to its facilitation of microglial activation induced by high glucose levels, a process mediated by the ROCK pathway. These findings highlight the importance of considering USP25 as a potential therapeutic target for the management of diabetic neuroinflammation.

Discovery and Optimization of Selective Brain-Penetrant EBP Inhibitors that Enhance Oligodendrocyte Formation.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

Mixed-Mode Mindfulness-based cognitive therapy for psychological resilience, Self Esteem and Stigma of patients with schizophrenia: a randomized controlled trial.

BACKGROUND: People with schizophrenia often face challenges such as lower psychological resilience, reduced self-worth, and increased social stigma, hindering their recovery. Mindfulness-Based Cognitive Therapy (MBCT) has shown promise in boosting psychological resilience and self-esteem while diminishing stigma. However, MBCT demands professional involvement and substantial expenses, adding to the workload of professionals and the financial strain on patients. Mixed-mode Mindfulness-Based Cognitive Therapy (M-MBCT) integrates both "face-to-face" and "self-help" approaches to minimize staff effort and costs. This study aims to assess the impact of M-MBCT on the psychological resilience, self-esteem, and stigma in schizophrenia patients. METHODS: This randomized, controlled, parallel-group, assessor-blinded clinical trial enrolled 174 inpatients with schizophrenia. Participants were randomly assigned to either the experimental or control group. The experimental group underwent an 8-week M-MBCT intervention, while the control group received standard treatment. Data collection employed the Connor-Davidson Resilience Scale (CD-RISC), Internalized Stigma of Mental Illness Scale (ISMI), and Rosenberg Self-Esteem Scale (RSES) before and after the intervention. Post-intervention, significant differences in ISMI, CD-RISC, and RSES scores were observed between the experimental and control groups. RESULTS: In the experimental group, ISMI scores notably decreased, while CD-RISC and RSES scores significantly increased (P < 0.05). Multiple linear regression analysis identified age, education, and family history of mental illness as significant factors related to stigma (P < 0.05). Additionally, correlation analysis indicated a significant negative relationship between the reduction in CD-RISC scores and the reduction in ISMI scores (P < 0.05). CONCLUSION: M-MBCT effectively enhanced psychological resilience and self-esteem while diminishing stigma in individuals with schizophrenia. M-MBCT emerges as a promising treatment option for schizophrenia sufferers. TRIAL REGISTRATION: The trial was registered at the Chinese Clinical Trial Registry on 03/06/2023 ( www.chictr.org.cn ; ChiCTR ID: ChiCTR2300069071).

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Structure- and machine learning-guided engineering demonstrate that a non-canonical disulfide in an anti-PD-1 rabbit antibody does not impede antibody developability.

Rabbits produce robust antibody responses and have unique features in their antibody repertoire that make them an attractive alternative to rodents for in vivo discovery. However, the frequent occurrence of a non-canonical disulfide bond between complementarity-determining region (CDR) H1 (C35a) and CDRH2 (C50) is often seen as a liability for therapeutic antibody development, despite limited reports of its effect on antibody binding, function, and stability. Here, we describe the discovery and humanization of a human-mouse cross-reactive anti-programmed cell death 1 (PD-1) monoclonal rabbit antibody, termed h1340.CC, which possesses this non-canonical disulfide bond. Initial removal of the non-canonical disulfide resulted in a loss of PD-1 affinity and cross-reactivity, which led us to explore protein engineering approaches to recover these. First, guided by the sequence of a related clone and the crystal structure of h1340.CC in complex with PD-1, we generated variant h1340.SA.LV with a potency and cross-reactivity similar to h1340.CC, but only partially recovered affinity. Side-by-side developability assessment of both h1340.CC and h1340.SA.LV indicate that they possess similar, favorable properties. Next, and prompted by recent developments in machine learning (ML)-guided protein engineering, we used an unbiased ML- and structure-guided approach to rapidly and efficiently generate a different variant with recovered affinity. Our case study thus indicates that, while the non-canonical inter-CDR disulfide bond found in rabbit antibodies does not necessarily constitute an obstacle to therapeutic antibody development, combining structure- and ML-guided approaches can provide a fast and efficient way to improve antibody properties and remove potential liabilities.

Mnox Nanoenzyme Armed CAR-NK Cells Enhance Solid Tumor Immunotherapy by Alleviating the Immunosuppressive Microenvironment.

Adoptively transferred cells usually suffer from exhaustion, limited expansion, and poor infiltration, partially attributing to the complicated immunosuppressive microenvironment of solid tumors. Therefore, it is necessary to explore more effective strategies to improve the poor tumor microenvironment (TME) to efficaciously deliver and support extrinsic effector cells in vivo. Herein, an intelligent biodegradable hollow manganese dioxide nanoparticle (MnOX) that possesses peroxidase activity to catalyze excess H2O2 in the TME to produce oxygen and relieve the hypoxia of solid tumors is developed. MnOX nanoenzymes modified with CD56 antibody could specifically bind CAR-NK (chimeric antigen receptor modified natural killer) cells. It is demonstrated that CAR-NK cells incorporated with MnOX nanoenzymes effectively infiltrate into tumor tissues with an improved TME, which results in superior antitumor activity in solid tumor-bearing mice. The antibody connection between MnOX nanoenzymes and CAR-NK endows the lowest efficient dosage of MnOX. This study features a smart synergistic immunotherapy approach for solid tumors using MnOX nanoenzyme-armed CAR-NK cells, which would provide a valuable tool for immunocyte therapy in solid tumors.

Role of Interleukin-21 in retinal ischemia-reperfusion injury: Unveiling the impact on retinal ganglion cell apoptosis.

BACKGROUND: Retinal ischemia-reperfusion (I/R) serves as a significant contributor to ocular diseases, triggering a cascade of pathological processes. The interplay between neuroinflammation and the apoptosis of retinal ganglion cell (RGC) is a well-explored aspect of retinal I/R-induced tissue damage. Within this intricate landscape, the inflammatory cytokine Interleukin-21 (IL21) emerges as a potent mediator of neuroinflammation with known detrimental effects on neuronal integrity. However, its specific impact on RGC apoptosis in the context of retinal I/R has remains to be uncovered. This study aims to unravel the potential anti-apoptotic effects of IL21 siRNA on RGC, shedding light on the neuroprotection of retinal I/R. METHODS: Sprague-Dawley (SD) rats underwent a controlled elevation of intraocular pressure (IOP) to 110 mmHg for 60 min to simulate retinal I/R conditions. To explore the influence of IL21 on RGC apoptosis and its underlying molecular mechanisms, a comprehensive array of techniques such immunohistochemistry, immunofluorescence, TUNEL, Hematoxylin-eosin (H&E), immunoblotting, and qRT-PCR were carried out. RESULTS: The landscape of retinal I/R injury revealed an increase in the expression of IL21, reaching its peak at 72 h. Notably, IL21 markedly induced RGC apoptosis within the retinal I/R milieu. The introduction of IL21 siRNA showed promising outcomes, manifesting as an amelioration of neurological function deficits, a reduction in RGC loss, and an increase in the thickness of the inner retinal layer at the 72-hour reperfusion. Additionally, IL21 siRNA demonstrated its ability to hinder the release of proteins associated with apoptosis via the JAK/STAT signaling pathway. In the in vitro setting, IL21 siRNA efficiently reduced R28 cell apoptosis by suppressing the production of proteins associated with apoptosis by regulating the JAK/STAT signaling pathway. CONCLUSIONS: This study provides evidence for the pathogenic role of IL21 in retinal I/R. The findings underscore IL21 siRNA as a promising therapeutic target for ischemic retinal injury. Its efficacy lies in its ability to mitigate RGC apoptosis by suppressing the JAK/STAT signaling pathway. These findings not only enhance our comprehension of retinal I/R pathology but also suggests IL21 siRNA as a potential transformative factor in the development of targeted therapies for ischemic retinal injuries.