STM2457 Inhibits METTL3-Mediated m6A Modification of miR-30c to Alleviate Spinal Cord Injury by Inducing the ATG5-Mediated Autophagy.

OBJECTIVE: The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins. METHODS: An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs. RESULTS: In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test. CONCLUSION: STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.

Microenvironment-responsive, multimodulated herbal polysaccharide hydrogel for diabetic foot ulcer healing.

Diabetic ulcers (DUs) usually suffer from severe infections, persistent inflammation, and excessive oxidative stress during the healing process, which led to the microenvironmental alternation and severely impede DU healing, resulting in a delayed wound healing. Therefore, it is particularly important to develop a medical dressing that can address these problems simultaneously. To this end, self-healing composite hydrogels were prepared in this study utilizing Bletilla striata polysaccharide (BSP) and Berberine (BER) with borax via borate ester bond. The chemical and mechanical properties of the BSP/BER hydrogels were characterized, and their wound healing performance was investigated in vivo and in vitro. The results showed that the BSP/BER hydrogel significantly accelerated wound healing in DU mice with the healing rate of 94.90 ± 1.81% on the 14th day by using BSP/BER5, and this outstanding performance was achieved by the multi-targeted biological functions of antibacterial, anti-inflammatory and antioxidant, which provided favorable microenvironment for orderly recovery of the wound. Aside from exhibiting the antibacterial rate of over 90% against both Escherichia coli and Staphylococcus aureus, the BSP/BER5 hydrogel could significantly reduce NO levels 4.544 ± 0.32 µmol/L to exert its anti-inflammatory effects. Additionally, it demonstrated a hemolysis rate and promotes cell migration capabilities at (34.92 ± 1.66%). With the above features, the developed BSP/BER hydrogel in this study could be the potential dressing for clinical treatment of DU wound.

Modeling "Two-Hit" Severe Pneumonia in Mice: Pathological Characteristics and Mechanistic Studies.

Severe pneumonia is one of the most common critical diseases in clinical practice. Existing models for severe pneumonia have limitations, leading to limited clinical translation. In this study, a two-hit severe pneumonia mouse model was established by inducing primary pneumonia through intratracheal instillation of 800 µg lipopolysaccharide (LPS), followed by intraperitoneal injection of 10 mg/kg LPS. The effectiveness of various inflammatory indicators and the lung tissue damage during the time course of this model were confirmed and evaluated. At 3 h post two-hit, the IL-6, TNF-α levels in peripheral blood and bronchoalveolar lavage fluid (BALF), and the white blood cells, neutrophils, and lymphocytes in BALF notably exhibited the most pronounced elevation. At 12 h post two-hit, the white blood cells and neutrophils in peripheral blood significantly increased, accompanied by notable alterations in splenic immune cells and worsened pulmonary histopathological damage. Transcriptomics of lung tissue, microbiota analysis of lung and gut, as well as plasma metabolomics analyses further indicated changes in transcriptional profiles, microbial composition, and metabolites due to the two-hit modeling. These results validate that the two-hit model mimics the clinical presentation of severe pneumonia and serves as a robust experimental tool for studying the pathogenesis of severe pneumonia and developing and assessing treatment strategies.

Are the facet joint parameters risk factors for cage subsidence after TLIF in patients with lumbar degenerative spondylolisthesis?

PURPOSE: To assess whether preoperative facet joint parameters in patients with degenerative lumbar spondylolisthesis (DS) are risk factors for cage subsidence (CS) following transforaminal lumbar interbody fusion (TLIF). METHODS: We enrolled 112 patients with L4-5 DS who underwent TLIF and were followed up for > 1 year. Preoperative demographic characteristics, functional areas of paraspinal muscles and psoas major muscles (PS), total functional area relative to vertebral body area, functional cross-sectional area (FCSA) of PS and lumbar spine extensor muscles, normalized FCSA of PS to the vertebral body area (FCSA/VBA), lumbar indentation value, facet joint orientation, facet joint tropism (FT), cross-sectional area of the superior articular process (SAPA), intervertebral height index, vertebral Hounsfield unit (HU) value, lordosis distribution index, t-scores, sagittal plane parameters, visual analog scale (VAS) for low back pain, VAS for leg pain, Oswestry disability index, global alignment and proportion score and European quality of life-5 dimensions (EQ-5D) were assessed. RESULTS: Postoperative CS showed significant correlations with preoperative FO(L3-4), FT (L3 and L5), SAPA(L3-5), L5-HU, FCSA/VBA(L3-4), Pre- T-score, post-6-month VAS for back pain and EQ-5D scores among other factors. According to ROC curve analysis, the optimal decision points for FO(L3-4), L3-SAPA, FCSA/VBA(L3-4), L5-HU, and Pre- T-score were 35.88°, 43.76°,114.93, 1.73, 1.55, 136, and - 2.49. CONCLUSIONS: This study identified preoperative FO, SAPA, preoperative CT, Pre- T-score and the FCSA/VBA as independent risk factors for CS after TLIF for DS. These risk factors should enable spinal surgeons to closely monitor and prevent the occurrence of CS.

Age and sex differences in emotion perception are influenced by emotional category and communication channel.

Sex differences in verbal and nonverbal emotion processing in older individuals are underexplored despite declining emotional performance with age. This study aimed to investigate the nature of sex differences in age-related decline in emotion perception, exploring modulatory effects on communication channels and emotion categories. Seventy-three older adults (43 female participants, aged 60-89 years) and 74 younger adults (37 female participants, aged 18-30 years) completed a task to recognize basic emotions (i.e., anger, happiness, neutrality, sadness) expressed by female or male encoders through verbal (i.e., semantic) and nonverbal (i.e., facial and prosodic) channels. Female participants consistently demonstrated an overall advantage in emotion perception and expression across both age cohorts. In older adults, this superiority was heightened in decoding angry and sad faces, as well as angry prosody and happy and sad semantics. However, older individuals exhibited decreased sensitivities to angry semantics, sad prosody, and neutral prosody from female encoders, whereas they showed heightened sensitivities to happy faces from female encoders and angry faces from male encoders. Both older and younger adults displayed age-related changes in sex interactions specific to emotional categories and channels. But neither own-sex nor opposite-sex bias was systematically observed across the two age groups. These results suggest that explicit emotion processing involves an intricate integration of individual and contextual differences, with significant age and sex interplay linked to specific emotions and channels. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Bletilla striata Polysaccharide-/Chitosan-Based Self-Healing Hydrogel with Enhanced Photothermal Effect for Rapid Healing of Diabetic Infected Wounds via the Regulation of Microenvironment.

The management of diabetic ulcers poses a significant challenge worldwide, and persistent hyperglycemia makes patients susceptible to bacterial infections. Unfortunately, the overuse of antibiotics may lead to drug resistance and prolonged infections, contributing to chronic inflammation and hindering the healing process. To address these issues, a photothermal therapy technique was incorporated in the preparation of wound dressings. This innovative solution involved the formulation of a self-healing and injectable hydrogel matrix based on the Schiff base structure formed between the oxidized Bletilla striata polysaccharide (BSP) and hydroxypropyltrimethylammonium chloride chitosan. Furthermore, the introduction of CuO nanoparticles encapsulated in polydopamine imparted excellent photothermal properties to the hydrogel, which promoted the release of berberine (BER) loaded on the nanoparticles and boosted the antibacterial performance. In addition to providing a reliable physical protection to the wound, the developed hydrogel, which integrated the herbal components of BSP and BER, effectively accelerated wound closure via microenvironment regulation, including alleviated inflammatory reaction, stimulated re-epithelialization, and reduced oxidative stress based on the promising results from cell and animal experiments. These impressive outcomes highlighted their clinical potential in safeguarding the wound against bacterial intrusion and managing diabetic ulcers.

Robustness of response-adaptive randomization.

Doubly adaptive biased coin design (DBCD), a response-adaptive randomization scheme, aims to skew subject assignment probabilities based on accrued responses for ethical considerations. Recent years have seen substantial advances in understanding DBCD's theoretical properties, assuming correct model specification for the responses. However, concerns have been raised about the impact of model misspecification on its design and analysis. In this paper, we assess the robustness to both design model misspecification and analysis model misspecification under DBCD. On one hand, we confirm that the consistency and asymptotic normality of the allocation proportions can be preserved, even when the responses follow a distribution other than the one imposed by the design model during the implementation of DBCD. On the other hand, we extensively investigate three commonly used linear regression models for estimating and inferring the treatment effect, namely difference-in-means, analysis of covariance (ANCOVA) I, and ANCOVA II. By allowing these regression models to be arbitrarily misspecified, thereby not reflecting the true data generating process, we derive the consistency and asymptotic normality of the treatment effect estimators evaluated from the three models. The asymptotic properties show that the ANCOVA II model, which takes covariate-by-treatment interaction terms into account, yields the most efficient estimator. These results can provide theoretical support for using DBCD in scenarios involving model misspecification, thereby promoting the widespread application of this randomization procedure.

FOXP4-mediated induction of PTK7 activates the Wnt/ß-catenin pathway and promotes ovarian cancer development.

Ovarian cancer (OV) poses a significant challenge in clinical settings due to its difficulty in early diagnosis and treatment resistance. FOXP4, belonging to the FOXP subfamily, plays a pivotal role in various biological processes including cancer, cell cycle regulation, and embryonic development. However, the specific role and importance of FOXP4 in OV have remained unclear. Our research showed that FOXP4 is highly expressed in OV tissues, with its elevated levels correlating with poor prognosis. We further explored FOXP4's function through RNA sequencing and functional analysis in FOXP4-deficient cells, revealing its critical role in activating the Wnt signaling pathway. This activation exacerbates the malignant phenotype in OV. Mechanistically, FOXP4 directly induces the expression of protein tyrosine kinase 7 (PTK7), a Wnt-binding receptor tyrosine pseudokinase, which causes abnormal activation of the Wnt signaling pathway. Disrupting the FOXP4-Wnt feedback loop by inactivating the Wnt signaling pathway or reducing FOXP4 expression resulted in the reduction of the malignant phenotype of OV cells, while restoring PTK7 expression reversed this effect. In conclusion, our findings underscore the significance of the FOXP4-induced Wnt pathway activation in OV, suggesting the therapeutic potential of targeting this pathway in OV treatment.

CXXC5 drove inflammation and ovarian cancer proliferation via transcriptional activation of ZNF143 and EGR1.

CXXC5, a zinc-finger protein, is known for its role in epigenetic regulation via binding to unmethylated CpG islands in gene promoters. As a transcription factor and epigenetic regulator, CXXC5 modulates various signaling processes and acts as a key coordinator. Altered expression or activity of CXXC5 has been linked to various pathological conditions, including tumorigenesis. Despite its known role in cancer, CXXC5's function and mechanism in ovarian cancer are unclear. We analyzed multiple public databases and found that CXXC5 is highly expressed in ovarian cancer, with high expression correlating with poor patient prognosis. We show that CXXC5 expression is regulated by oxygen concentration and is a direct target of HIF1A. CXXC5 is critical for maintaining the proliferative potential of ovarian cancer cells, with knockdown decreasing and overexpression increasing cell proliferation. Loss of CXXC5 led to inactivation of multiple inflammatory signaling pathways, while overexpression activated these pathways. Through in vitro and in vivo experiments, we confirmed ZNF143 and EGR1 as downstream transcription factors of CXXC5, mediating its proliferative potential in ovarian cancer. Our findings suggest that the CXXC5-ZNF143/EGR1 axis forms a network driving ovarian cell proliferation and tumorigenesis, and highlight CXXC5 as a potential therapeutic target for ovarian cancer treatment.

Structure of a polysaccharide MDP2-1 from Melastoma dodecandrum Lour. and its anti-inflammatory effects.

The anti-inflammatory activity of polysaccharides derived from Melastoma dodecandrum Lour. was evaluated in pyretic mice and HEK-Blue™ hTLR4 cells. The testing led to the identification of MDP2-1, which was then investigated for its structural characteristics and anti-inflammatory effects. Results showed that MDP2-1 had a molecular weight of 29.234 kDa and primarily consisted of galactose, arabinose, rhamnose, glucose, glucuronic acid, and galacturonic acid. Its main backbone was composed of →4)-α-D-GalpA-(1→, →2)-α-L-Rhap-(1→, →3,4)-α-D-GalpA-(1→, →2,4)-α-D-GlcpA-(1→, and its side chains were connected by →4)-α-D-Galp-(1→, α-D-Galp-(1→, →4)-ß-D-Glcp-(1→, and α-L-Araf-(1→. In vivo experiments on mice demonstrated that MDP2-1 attenuated LPS-induced acute lung injury, and in vitro experiments on RAW264.7 cells showed that MDP2-1 reduced the levels of inflammatory mediators and mitigated LPS-induced inflammatory damage by inhibiting the activation of the TLR4 downstream NF-κB/MAPK pathway. These findings suggest that MDP2-1 is a novel anti-inflammatory agent for therapeutic interventions.

Purple Urine Bag Syndrome Amelioration without Antibiotic Therapy.

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Effects of pharmaceutical and personal care products on pubertal development: Evidence from human and animal studies.

Pharmaceutical and personal care products (PPCPs) include a wide range of drugs, personal care products and household chemicals that are produced and used in significant quantities. The safety of PPCPs has become a growing concern in recent decades due to their ubiquitous presence in the environment and potential risks to human health. PPCPs have been detected in various human biological samples, including those from children and adolescents, at concentrations ranging from several ng/L to several thousand µg/L. Epidemiological studies have shown associations between exposure to PPCPs and changes in the timing of puberty in children and adolescents. Animal studies have shown that exposure to PPCPs results in advanced or delayed pubertal onset. Mechanisms by which PPCPs regulate pubertal development include alteration of the hypothalamic kisspeptin and GnRH networks, disruption of steroid hormones, and modulation of metabolic function and epigenetics. Gaps in knowledge and further research needs include the assessment of environmental exposure to pharmaceuticals in children and adolescents, low-dose and long-term effects of exposure to PPCPs, and the modes of action of PPCPs on pubertal development. In summary, this comprehensive review examines the potential effects of exposure to PPCPs on pubertal development based on evidence from human and animal studies.

Bletilla striata polysaccharides protect against ARDS by modulating the NLRP3/caspase1/GSDMD and HMGB1/TLR4 signaling pathways to improve pulmonary alveolar macrophage pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata polysaccharides (BSP) extracted from the B. striata tuber, have been demonstrated to possess anti-inflammatory properties. However, their potential protective effect against ARDS and their role in regulating cell pyroptosis remained unexplored. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic effect of BSP in the alleviation of lipopolysaccharide (LPS)-induced ARDS, and to explore its mechanism of action. METHODS: The effect of BSP was assessed by LPS injection into the intraperitoneal cavity in vivo; pathological changes of ARDS mice were gauged by immunohistochemical, hematoxylin and eosin staining, and immunofluorescence assays. MH-S cells were used to model the pyroptosis in vitro. Finally, the pyroptosis of alveolar macrophage was detected by western blots, qPCR, and flow cytometry for NLRP3/caspase1/GSDMD and HMGB1/TLR4 pathway-associated proteins and mRNA. RESULTS: BSP could significantly increase the weight and survival rate of mice with ARDS, alleviate the cytokine storm in the lungs, and reduce lung damage in vivo. BSP inhibited the inflammation caused by LPS/Nigericin significantly in vitro. Compared with the control group, there was a remarkable surge in the incidence of pyroptosis observed in ARDS lung tissue and alveolar macrophages, whereas BSP significantly diminished the pyroptosis ratio. Besides, BSP reduced NLRP3/caspase1/GSDMD and HMGB1/TLR4 levels in ARDS lung tissue and MH-S cells. CONCLUSIONS: These findings proved that BSP could improve LPS-induced ARDS via inhibiting pyroptosis, and this effect was mediated by NLRP3/caspase1/GSDMD and HMGB1/TLR4, suggesting a therapeutic potential of BSP as an anti-inflammatory agent for ARDS treatment.

Triclosan induces earlier puberty onset in female mice via interfering with L-type calcium channels and activating Pik3cd.

Triclosan (TCS) is a broad-spectrum antibacterial chemical widely presents in people's daily lives. Epidemiological studies have revealed that TCS exposure may affect female puberty development. However, the developmental toxicity after low-dose TCS continuous exposure remains to be confirmed. In our study, 8-week-old ICR female mice were continuously exposed to TCS (30, 300, 3000 µg/kg/day) or vehicle (corn oil) from 2 weeks before mating to postnatal day 21 (PND 21) of F1 female mice, while F1 female mice were treated with TCS intragastric administration from PND 22 until PND 56. Vaginal opening (VO) observation, hypothalamic-pituitary-ovarian (HPO) axis related hormones and genes detection, and ovarian transcriptome analysis were carried out to investigate the effects of TCS exposure on puberty onset. Meanwhile, human granulosa-like tumor cell lines (KGN cells) were exposed to TCS to further explore the biological mechanism of the ovary in vitro. The results showed that long-term exposure to low-dose TCS led to approximately a 3-day earlier puberty onset in F1 female mice. Moreover, TCS up-regulated the secretion of estradiol (E2) and the expression of ovarian steroidogenesis genes. Notably, ovarian transcriptomes analysis as well as bidirectional validation in KGN cells suggested that L-type calcium channels and Pik3cd were involved in TCS-induced up-regulation of ovarian-related hormones and genes. In conclusion, our study demonstrated that TCS interfered with L-type calcium channels and activated Pik3cd to up-regulate the expression of ovarian steroidogenesis and related genes, thereby inducing the earlier puberty onset in F1 female mice.

Modified FGF Hydrogel for Effective Axon Formation by Enhanced Regeneration of Myelin Sheath of Schwann Cells Using Rat Model.

Introduction: An acute spinal cord injury (SCI) is a debilitating event for which there is no targeted or effective treatment. Previous studies have shown that fibroblast growth factor (bFGF) and Schwann cells (SC) exert a protective effect on the injured tissues. Because of their easy injectability and strength, hydrogels are considered to be ideal candidates for creating loadable tissues. However, the application and mechanism of bFGF-hydrogels have not been explored. Methods: We synthesized a new class of bFGF-hydrosol and evaluated its safety and biocompatibility in vitro and in vivo. Next, an SCI rat model was established to evaluate the effect of the hydrosol on an SCI by detecting various pro-inflammatory markers and evaluating the injury. The ability of hydrosol to promote axon formation was evaluated by detecting corresponding indexes, and its ability to promote remyelination was evaluated by detecting the corresponding indexes in Schwann cells. Results: A novel in situ injectable hydrogel containing bFGF (HA-bFGF) was synthesized and found to have better biocompatibility than other gels. HA-bFGF helped to repair tissue damage after an SCI in vivo. Our mechanistic investigation also showed that HA-bFGF improved axon formation after an SCI by facilitating the regeneration of myelin sheath of Schwann cells. Conclusion: In this study, we found that HA-bFGF could promote neural restoration and tissue recovery after an SCI. Our results indicate that hydrogels loaded with bFGF can alleviate a spinal cord injury by promoting the remyelination of Schwann cells, reducing inflammation at the injured site, and ultimately promoting axon generation.

A Berberine-Loaded Bletilla striata Polysaccharide Hydrogel as a New Medical Dressing for Diabetic Wound Healing.

The healing process of a diabetic wound (DW) is often impeded by a series of interrelated factors, including severe infection, persistent inflammation, and excessive oxidative stress. Therefore, it is particularly crucial to develop a medical dressing that can address these issues simultaneously. To this end, different ratios of Bletilla striata polysaccharide (BSP) and berberine (BER) were physically blended with Carbomer 940 (CBM940) to develop a composite hydrogel as a medical dressing. The BSP/BER hydrogel was characterized using SEM, FTIR, rheological testing and other techniques. The anti-inflammatory, antioxidant, and antibacterial properties of the hydrogel were evaluated using cell and bacterial models in vitro. A DW model of ICR mice was established to evaluate the effect of the hydrogel on DW healing in vivo. The hydrogel exhibited excellent biocompatibility and remarkable antibacterial, anti-inflammatory, and antioxidant properties. In addition, animal experiments showed that the BSP/BER hydrogel significantly accelerated wound healing in DW mice. Among the different formulations, the LBSP/BER hydrogel (2% BSP, mBER:mBSP = 1:40) demonstrated the most remarkable efficacy. In conclusion, the BSP/BER hydrogel developed exhibited immense properties and great potential as a medical dressing for the repair of DW, addressing a crucial need in clinical practice.

Association between exposure to persistent organic pollutants and pubertal timing in boys and girls: A systematic review and meta-analysis.

In recent years, the phenomenon of abnormal pubertal timing in children has become increasingly common worldwide. Persistent organic pollutants (POPs) may be one of the risk factors contributing to this phenomenon, but the relationship between them is unclear based on current evidence. The purpose of this study was to determine the association of POPs exposure with pubertal timing in girls and boys by conducting a systematic review and meta-analysis. We searched PubMed and Embase databases for studies before June 1, 2023. Meta-analysis was performed by pooling relative risk (RR) or odds ratio (OR) or prevalence ratio (PR) or hazard ratio (HR) estimates with 95 % confidence intervals (CIs). Subgroup analysis, publication bias assessment and sensitivity analysis were also carried out. A total of 21 studies were included, involving 2479 boys and 8718 girls. The results of meta-analysis showed that exposure to POPs was significantly associated with delayed pubertal timing in girls (RR: 0.85; 95 % CI: 0.79-0.91; p < 0.001). There was no statistically significant association between exposure to POPs and pubertal timing in boys (RR: 1.18; 95 % CI: 0.99-1.40; p = 0.070). Subgroup analysis showed that there may be gender differences in the effects of exposure to POPs on pubertal timing. Our results suggested that exposure to POPs could delay pubertal timing in girls. However, based on current evidence, no significant association was found between POPs exposure and pubertal timing in boys.

Preparation strategy of hydrogel microsphere and its application in skin repair.

In recent years, hydrogel microsphere has attracted much attention due to its great potential in the field of skin repair. This paper reviewed the recent progress in the preparation strategy of hydrogel microsphere and its application in skin repair. In this review, several preparation methods of hydrogel microsphere were summarized in detail. In addition, the related research progress of hydrogel microspheres for skin repair was reviewed, and focused on the application of bioactive microspheres, antibacterial microspheres, hemostatic microspheres, and hydrogel microspheres as delivery platforms (hydrogel microspheres as a microcarrier of drugs, bioactive factors, or cells) in the field of skin repair. Finally, the limitations and future prospects of the development of hydrogel microspheres and its application in the field of skin repair were presented. It is hoped that this review can provide a valuable reference for the development of the preparation strategy of hydrogel microspheres and promote the application of hydrogel microspheres in skin repair.

Dimer Peptide Ligands of Vascular Endothelial Growth Factor: Optimizing Linker Length for High Affinity and Antiangiogenic Activity.

Macromolecular ligands targeting vascular endothelial growth factor A (VEGF) to inhibit pathological angiogenesis are used in the clinic for the treatment of cancers and ocular diseases. To develop smaller ligands retaining high affinity through an avidity effect, here we design homodimer peptides targeting the two symmetrical binding sites of the VEGF homodimer. A series of 11 dimers were synthesized with flexible poly(ethylene glycol) (PEG) linkers of increasing lengths. The binding mode was determined by size exclusion chromatography, and analytical thermodynamic parameters were measured by isothermal titration calorimetry and compared to the antibody bevacizumab. The effect of linker length was qualitatively correlated to a theoretical model. With the optimal length in PEG25-dimer D6, the binding affinity was improved 40-fold compared to a monomer control, resulting in a single-digit nanomolar Kd value. Finally, we validated the benefit of the dimerization strategy by evaluating the activity of control monomers and selected dimers in cell-based assays with human umbilical vein endothelial cells (HUVECs).