MB2033, an anti-PD-L1 × IL-2 variant fusion protein, demonstrates robust anti-tumor efficacy with minimal peripheral toxicity.

Interleukin-2 (IL-2), a cytokine with pleiotropic immune effects, was the first approved cancer immunotherapy agent. However, IL-2 is associated with systemic toxicity due to binding with its ligand IL-2Rα, such as vascular leakage syndrome, limiting its clinical applications. Despite efforts to extend the half-life of IL-2 and abolish IL-2Rα interactions, the risk of toxicity remains unresolved. In this study, we developed the bispecific fusion protein MB2033, comprising a novel IL-2 variant (IL-2v) connected to anti-programmed death ligand 1 (PD-L1) via a silenced Fc domain. The IL-2v of MB2033 exhibits attenuated affinity for IL-2Rßγ without binding to IL-2Rα. The binding affinity of MB2033 for PD-L1 is greater than that for IL-2Rßγ, indicating its preferential targeting of PD-L1+ tumor cells to induce tumor-specific immune activation. Accordingly, MB2033 exhibited significantly reduced regulatory T cell activation, while inducing comparable CD8+ T cell activation to recombinant human IL-2 (rhIL-2). MB2033 induced lower immune cell expansion and reduced cytokine levels compared with rhIL-2 in human peripheral blood mononuclear cells, indicating a decreased risk of peripheral toxicity. MB2033 exhibited superior anti-tumor efficacy, including tumor growth inhibition and complete responses, compared with avelumab monotherapy in an MC38 syngeneic mouse model. In normal mice, MB2033 was safer than non-α IL-2v and tolerable up to 30 mg/kg. These preclinical results provide evidence of the dual advantages of MB2033 with an enhanced safety and potent clinical efficacy for cancer treatment.

Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection.

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

Antibacterial Photodynamic Inactivation of Fagopyrin F from Tartary Buckwheat (Fagopyrum tataricum) Flower against Streptococcus mutans and Its Biofilm.

The objective of this study was to determine reactive oxygen species (ROS) produced by fagopyrin F-rich fraction (FFF) separated from Tartary buckwheat flower extract exposed to lights and to investigate its antibacterial photodynamic inactivation (PDI) against Streptococcus mutans and its biofilm. ROS producing mechanisms involving FFF with light exposure were determined using a spectrophotometer and a fluorometer. S. mutans and its biofilm inactivation after PDI treatment of FFF using blue light (BL; 450 nm) were determined by plate count method and crystal violet assay, respectively. The biofilm destruction by ROS produced from FFF after exposure to BL was visualized using confocal laser scanning microscopy (CLSM) and field emission scanning electron microscope (FE-SEM). BL among 3 light sources produced type 1 ROS the most when applying FFF as a photosensitizer. FFF exposed to BL (5 and 10 J/cm2) significantly more inhibited S. mutans viability and biofilm formation than FFF without the light exposure (p < 0.05). In the PDI of FFF exposed to BL (10 J/cm2), an apparent destruction of S. mutans and its biofilm were observed by the CLSM and FE-SEM. Antibacterial PDI effect of FFF was determined for the first time in this study.

Substance P enhances the therapeutic effect of MSCs by modulating their angiogenic potential.

Bone marrow mesenchymal stem cell (MSC) therapy acts through multiple differentiations in damaged tissue or via secretion of paracrine factors, as demonstrated in various inflammatory and ischaemic diseases. However, long-term ex vivo culture to obtain a sufficient number of cells in MSC transplantation leads to cellular senescence, deficiency of the paracrine potential, and loss of survival rate post-transplantation. In this study, we evaluated whether supplementation of MSCs with substance P (SP) can improve their therapeutic potential. SP treatment elevated the secretion of paracrine/angiogenic factors, including VEGF, SDF-1a and PDGF-BB, from late passage MSCs in vitro. MSCs supplemented with SP accelerated epidermal/dermal regeneration and neovascularization and suppressed inflammation in vivo, compared to MSCs transplanted alone. Importantly, supplementation with SP enabled the incorporation of transplanted human MSCs into the host vasculature as pericytes via PDGF signalling, leading to the direct engagement of transplanted cells in compact vasculature formation. Our results showed that SP is capable of restoring the cellular potential of senescent stem cells, possibly by modulating the generation of paracrine factors from MSCs, which might accelerate MSC-mediated tissue repair. Thus, SP is anticipated to be a potential beneficial agent in MSC therapy for inflammatory or ischaemic diseases and cutaneous wounds.

Phytoene synthase 2 can compensate for the absence of PSY1 in the control of color in Capsicum fruit.

Phytoene synthase 1 (PSY1) and capsanthin-capsorubin synthase (CCS) are two major genes responsible for fruit color variation in pepper (Capsicum spp.). However, the role of PSY2 remains unknown. We used a systemic approach to examine the genetic factors responsible for the yellow fruit color of C. annuum 'MicroPep Yellow' (MY) and to determine the role of PSY2 in fruit color. We detected complete deletion of PSY1 and a retrotransposon insertion in CCS. Despite the loss of PSY1 and CCS function, both MY and mutant F2 plants from a cross between MY and the 'MicroPep Red' (MR) accumulated basal levels of carotenoids, indicating that other PSY genes may complement the loss of PSY1. qRT-PCR analysis indicated that PSY2 was constitutively expressed in both MR and MY fruits, and a color complementation assay using Escherichia coli revealed that PSY2 was capable of biosynthesizing a carotenoid. Virus-induced gene silencing of PSY2 in MY resulted in white fruits. These findings indicate that PSY2 can compensate for the absence of PSY1 in pepper fruit, resulting in the yellow color of MY fruits.

Relationship of breastfeeding duration with joint pain and knee osteoarthritis in middle-aged Korean women: a cross-sectional study using the Korea National Health and Nutrition Examination Survey.

BACKGROUND: The effect of joint health on the quality of life of middle-aged and older women is becoming more widely recognized with the aging of the world's population. However, the association of long-term breastfeeding with joint pain and knee osteoarthritis has not been fully examined. The aim of this study was to determine the association of prior breastfeeding duration with current joint pain and knee osteoarthritis in middle-aged Korean women. METHODS: This cross-sectional study was conducted among 3454 women aged ≥50 years who underwent knee radiography and answered a questionnaire on breastfeeding and joint pain for the 5th Korea National Health and Nutrition Examination Survey (2010-2011). After adjusting for confounding sociodemographic, medical history, and obstetric and gynecologic variables, logistic regression analysis was conducted to analyze the prevalence of joint pain and knee osteoarthritis according to breastfeeding and its duration. RESULTS: Among the 3454 participants, 298 had not breastfed and 1042, 815, and 1299 had breastfed for 1-24, 25-48, and ≥ 49 months, respectively. Of all participants, 1731 had joint pain and 739 were diagnosed with knee osteoarthritis after radiography. Using the non-breastfeeding group as a reference, the odds ratio (OR) for joint pain among women who breastfed ≥1 month was 1.49 (95% confidence interval [CI] 1.01-2.21). As the breastfeeding duration increased, the OR of joint pain prevalence also increased (p for trend; p = 0.002). For knee osteoarthritis, the OR was 2.30 in the 25-48 months group (95% CI 1.09-4.86). The OR of knee osteoarthritis in the ≥49 months group was 2.17 (95% CI 1.01-4.64). Sensitivity analysis after selecting only participants aged ≥60 years showed that the prevalence of joint pain and knee osteoarthritis was more positively correlated with extended breastfeeding duration (joint pain, p for trend; p = 0.005) (knee osteoarthritis, p for trend; p = 0.012). CONCLUSIONS: Long-term feeding for more than 25 months was associated with an increased prevalence of joint pain and degenerative arthritis in Korean women aged ≥50 years.

Substance-P Prevents Cardiac Ischemia-Reperfusion Injury by Modulating Stem Cell Mobilization and Causing Early Suppression of Injury-Mediated Inflammation.

BACKGROUND/AIMS: Therapies using stem/progenitor cells have been experimentally and clinically investigated to regenerate damaged hearts. Substance-P (SP) induces bone marrow (BM) stem cell mobilization and suppresses inflammation in ischemic injuries. This study investigated the role of SP in BM stem cell mobilization and immune responses for tissue repair after ischemic-reperfusion injury (IRI), in comparison with that of granulocyte colony-stimulating factor (GCSF). METHODS: SP was intravenously injected into IRI rats and its affect was evaluated by determining colony forming efficiency, immune cell/ cytokine profiles, histological changes, and heart function through echocardiography. RESULTS: In the rat cardiac IRI model, SP suppressed IRI-mediated tumor necrosis factor-α induction, but increased the levels of interleukin-10, CD206+ monocytes, and regulatory T cells in the blood; reduced myocardial apoptosis at day 1 post-IRI; and markedly stimulated colony forming unit (CFU)-e and (CFU)-f cell mobilization. Efficacy of SP in the recovery of cardiac function after IRI was demonstrated by increased cardiac contractility, accompanied by reduced infarction sizes and fibrosis, and increased revascularization of vessels covered with alpha smooth muscle actin. These effects of SP were confirmed in an acute myocardial infarction (AMI) model. All effects mediated by SP were superior to those mediated by GCSF. CONCLUSION: Systemic injection of SP decreased early inflammatory responses and promoted stem cell mobilization, leading to a compact vasculature and improved cardiac function in cardiac IRI and AMI.

Single-molecule real-time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.).

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene ß-cyclase, ß-carotene hydroxylase, zeaxanthin epoxidase and capsanthin-capsorubin synthase (CCS) genes, in 94 pepper accessions by single-molecule real-time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra-performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture.

Substance P prevents development of proliferative vitreoretinopathy in mice by modulating TNF-α.

Purpose: Proliferative vitreoretinopathy (PVR) is an inflammatory fibrotic disease resulting from the inflammatory milieu after retinal detachment, which can prevent retinal healing. This study aimed to elucidate the effect of substance P (SP) on retinal degeneration caused by retinal detachment in vivo and to examine the role of SP in the tumor necrosis factor-alpha (TNF-α)-induced epithelial-mesenchymal transition (EMT) of human RPE cells in vitro. Methods: PVR-like retinal damage was induced by intravitreally injecting dispase into mice, and SP was systemically injected twice a week for 3 weeks. Histological analysis and cytokine profile with enzyme-linked immunosorbent assay (ELISA) were performed. The direct effect of SP on induction of EMT in vitro was studied by adding SP to TNF-α-treated ARPE-19 cells and then evaluating the change in the characteristics of the epithelial and mesenchymal cells. Results: Dispase injection led to a PVR-like retinal condition, demonstrating an inflammatory response with disruption of RPE interaction within 1 week and severe destruction with enfolding within 3 weeks after the dispase injection. The inflammatory environment promoted apoptosis and migration of fibroblast-like cells in the retinal layer, which can cause fibrotic disease, such as PVR. However, SP treatment suppressed early inflammatory responses by reducing TNF-α and elevating interleukin-10 (IL-10), with cell death and the appearance of fibroblastic cells inhibited and the progression of retinal degeneration obviously delayed. Moreover, SP ameliorated TNF-α-induced EMT of the RPE and directly prevented fibrotic change in the RPE. Conclusions: This study revealed that SP can block apoptosis and EMT due to retinal inflammation and inhibit the development of PVR. This effect most likely occurred by modulating the secretion and action of TNF-α..

Substance-P alleviates dextran sulfate sodium-induced intestinal damage by suppressing inflammation through enrichment of M2 macrophages and regulatory T cells.

Intestinal inflammation alters immune responses in the mucosa and destroys colon architecture, leading to serious diseases such as inflammatory bowel disease (IBD). Thus, regulation of inflammation is regarded as the ultimate therapy for intestinal disease. Substance-P (SP) is known to mediate proliferation, migration, and cellular senescence in a variety of cells. SP was found to mobilize stem cells from bone marrow to the site of injury and to suppress inflammatory responses by inducing regulatory T cells (Tregs) and M2 macrophages. In this study, we explored the effects of SP in a dextran sodium sulfate (DSS)-induced intestine damage model. The effects of SP were evaluated by analyzing crypt structures, proliferating cells within the colon, cytokine secretion profiles, and immune cells population in the spleen/mesenteric lymph nodes in vivo. DSS treatment provoked an inflammatory response with loss of crypts in the intestines of experimental mice. This response was associated with high levels of inflammatory cytokines such as TNF-α and IL-17, and low levels of Tregs and M2 macrophages, leading to severely damaged tissue structure. However, SP treatment inhibited inflammatory responses by modulating cytokine production as well as the balance of Tregs/Th 17 cells and the M1/M2 transition in lymphoid organs, leading to accelerated tissue repair. Collectively, our data indicate that SP can promote the regeneration of tissue following damage by DSS treatment, possibly by modulating immune response. Our results propose SP as a candidate therapeutic for intestine-related inflammatory diseases.

Substance P promotes diabetic wound healing by modulating inflammation and restoring cellular activity of mesenchymal stem cells.

Diabetic ulceration is one of the most debilitating complications of diabetes and is the main cause of amputation. The diabetic environment is characterized by prolonged inflammation and abnormal angiogenesis, leading to delayed wound healing. Thus, regulation of inflammation and neovascularization is considered a desirable target for diabetes. The critical purpose of this study was to determine whether systemically administered Substance P (SP) could promote wound healing in diabetic environments via suppression of inflammation, induction of angiogenesis, and mobilization of stem cells. The effect of SP was assessed by analyzing epidermal and dermal recovery, vessel formation, cytokine secretion profile, and the stem cell pool in the circulation and bone marrow. Compared with the vehicle-treated group, the SP-treated group exhibited more rapid wound coverage, reduced infiltration of leukocytes, suppression of injury-mediated enlargement of the spleen and mesenteric lymph nodes, reduced tumor necrosis factor-alpha levels, increased interleukin-10, elevated pool of M2 monocytes and vascular endothelial growth factor levels in the blood. Moreover, the stem cell pool in the bone marrow, which is very low in diabetes, was markedly restored by SP to normal levels, which could provide a favorable environment to facilitate wound healing in diabetes. This result demonstrates, for the first time, a possible application of SP for the treatment of diabetic complications, including diabetic ulcers.

Elevated cortisol content in dog hair with atopic dermatitis.

Canine atopic dermatitis (CAD) is a chronic relapsing inflammatory skin disease occurring in 10% of the canine population. Although most studies have focused on the pathophysiological mechanism involved in CAD, the detrimental impact of CAD on quality of life has received only little attention. Hair cortisol analysis is becoming a valuable tool in monitoring chronic stress. To further validate this approach in CAD, we compared the hair cortisol concentration of atopic dogs with that of healthy conditioned dogs. The extent and severity of cutaneous lesions of atopic dermatitis were assessed according to modified CADESI-03 scores. In addition, skin barrier function was evaluated by measuring transepidermal water loss (TEWL) and stratum corneum conductance. The correlation between CAD severity and hair cortisol concentration was evaluated. The level of hair cortisol evaluated by ELISA assay showed that the atopic dermatitis group had significantly increased cortisol levels compared to that of the healthy control group. A significant positive correlation was identified between hair cortisol level and the CADESI score in CAD patients. The TEWL value of the cubital flexor of the forelimb in the atopic group was significantly higher compared to the healthy controls. These findings imply that the hair cortisol analysis can be an effective and objective biomarker in assessment of long-term stress of CAD patients.

Effect of substance P on recovery from laser-induced retinal degeneration.

Retinal degeneration is caused by neovascularization and persistent inflammation in the retinal pigment epithelium (RPE) and choroid, and causes serious eye disease including age-related macular degeneration (AMD). Thus, inhibiting inflammation and neovascularization may be a primary approach to protect the retina from degeneration. The purpose of this study was to determine whether substance P (SP), which can suppress inflammation and mobilize stem cells, can protect the RPE from degeneration. The effect of SP was evaluated by analyzing systemic inflammation, cell survival, and neovascularization within the argon laser-injured retina of mice. At 1 week postinjury, the SP-treated group had lower tumor necrosis factor-alpha and higher interleukin-10 serum concentrations, and a more intact retinal structure compared to the vehicle-treated group. In mice administered SP repeatedly for 4 weeks, the retinal structure appeared normal and showed sparse neovascularization, whereas the vehicle-treated group showed severe retinal destruction and dense neovascularization. Moreover, the efficacy of SP was identical to that of mesenchymal stem cells that were transplanted into the vitreous after retinal injury. This study highlights the potential for the endogenous neuropeptide SP as a treatment for retinal damage to prevent conditions such as AMD.

Humidifier disinfectant-associated children's interstitial lung disease.

RATIONALE: Beginning in 2006, epidemics of a fatal lung injury of unknown cause in children were observed in Korea every spring. A recent study demonstrated that this type of children's interstitial lung disease (chILD) is associated with humidifier disinfectant use. OBJECTIVES: To determine the clinical characteristics of this type of chILD and to assess whether the nationwide suspension of humidifier disinfectant sales in the autumn of 2011 affected its incidence. METHODS: The clinical characteristics of suspected cases between 2006 and 2011 were determined by a nationwide retrospective study. The potential causal relationship with humidifier disinfectants was examined by a prospective surveillance study after humidifier disinfectant sales were suspended. MEASUREMENTS AND MAIN RESULTS: In total, 138 children were diagnosed with this type of chILD, which was characterized by rapid progression, high mortality, predominance in the spring season, and a familial tendency. The annual incidence increased in 2011 and then dropped to zero in 2012. The children were on average 30.4 months old. The most frequent symptoms at admission were cough and dyspnea. As the disease progressed, the typical complication was spontaneous air leak. Eighty children (58%) died. Two years after humidifier disinfectant-sale suspension, no more new cases were found. CONCLUSIONS: This study suggests that humidifier disinfectant inhalation causes an idiopathic type of chILD that is characterized by spontaneous air leak, rapid progression, lack of response to treatment, and high mortality. Further safety studies must be performed on common environmental compounds, particularly those that enter the human body by an unusual route.

Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component.

Rhus verniciflua Stokes has been used as a traditional herbal medicine in Asia. In this study, the effect of R. verniciflua extract on human aromatase (cytochrome P450 19, CYP19) activity was investigated to elucidate the mechanism for the effect of R. verniciflua extract on androgen hormone levels. Androstenedione was used as a substrate and incubated with R. verniciflua extract in cDNA-expressed CYP19 supersomes in the presence of NADPH, and estrone formation was measured using liquid chromatography-tandem mass spectrometry. R. verniciflua extract was assessed at concentrations of 10-1000 µg/mL. The resulting data showed that R. verniciflua extract inhibited CYP19-mediated estrone formation in a concentration-dependent manner with an IC50 value of 136 µg/mL. Subsequently, polyphenolic compounds from R. verniciflua extract were tested to identify the ingredients responsible for the aromatase inhibitory effects by R. verniciflua extract. As a result, butin showed aromatase inhibitory effect in a concentration-dependent manner with an IC50 value of 9.6 µM, whereas the inhibition by other compounds was negligible. These results suggest that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity and butin is a major ingredient responsible for this activity.

The inhibitory effect of Scutellaria baicalensis extract and its active compound, baicalin, on the translocation of the androgen receptor with implications for preventing androgenetic alopecia.

Androgens affect several human skin and prostate functions, and the androgen receptor is crucial for regulating the androgen-related mechanisms. In this study, we assessed the antagonizing effects of a Scutellaria baicalensis extract and its main component baicalin on proliferation of human scalp dermal papilla cells. First, the extract and baicalin slightly dissociated the radioisotope-labeled androgen receptor-agonist complex in the androgen receptor binding assay, and the IC50 values were measured to assess the androgen receptor antagonistic effect of the extract (93 µg/mL) and baicalin (54.1 µM). Second, the extract and baicalin treatments dose-dependently inhibited the overgrowth of LNCaP prostate cancer cells, which were stimulated by dihydrotestosterone. Third, the extract and baicalin inhibited nuclear translocation of the androgen receptor stimulated by dihydrotestosterone in human dermal papilla cells. Additionally, the extract and baicalin enhanced proliferation of human dermal papilla cells in vitro. These results show that the extract and baicalin inhibited androgen activation signaling and promoted hDPC proliferation, suggesting that they could be used as active ingredients for treating androgen-associated disorders, such as androgenetic alopecia.

Optimization of accelerated solvent extraction of zeaxanthin from orange paprika using response surface methodology and an artificial neural network coupled with a genetic algorithm.

This study aimed to optimize the accelerated solvent extraction (ASE) condition of zeaxanthin from orange paprika using a response surface methodology (RSM) or an artificial neural network (ANN) with a genetic algorithm (GA). Input variables were ethanol concentration, extraction time, and extraction temperature, while output variable was zeaxanthin. The mean squared error and regression correlation coefficient of the developed ANN model were 0.3038 and 0.9983, respectively. Predicted optimal extraction conditions from ANN-GA for maximum zeaxanthin were 100% ethanol, 3.4 min, and 99.2 °C. The relative errors under the optimal extraction conditions were RSM for 10.46% and ANN-GA for 2.18%. We showed that the recovery of hydrophobic zeaxanthin could be performed using ethanol, an eco-friendly solvent, via ASE, and the extraction efficiency could be improved by ANN-GA modeling than RSM. Therefore, combining ASE and ANN-GA might be desirable for the efficient and eco-friendly extraction of hydrophobic functional materials from food resources. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01514-8.

Correlation between aggression at the veterinary clinic and problem behaviors at home for cats in the USA.

OBJECTIVES: The aim of the study was to determine whether cats that exhibit aggression during veterinary visits are more likely to have behavior problems at home. METHODS: An online, anonymous, cross-sectional survey was developed and distributed to residents in the USA who were aged over 18 years and who were the primary owners of at least one cat. The survey collected information about cat and household factors, and utilized a validated questionnaire instrument for obtaining behavioral information of pet cats. RESULTS: Aggression at the veterinary clinic was reported in 42.6% of the cats. The frequency of aggression exhibited at the veterinary clinic was lower in cats that lived in multi-cat households. Most cats did not receive medications intended to reduce fear, anxiety and/or pain before veterinary visits. Aggression at the veterinary clinic was positively associated with behavior problems at home, including stranger-directed aggression, owner-directed aggression, resistance to restraint, familiar cat aggression, dog-directed aggression, house-soiling, separation-related behaviors and scratching claws on inappropriate surfaces indoors. CONCLUSIONS AND RELEVANCE: Cats that exhibit aggression at the veterinary clinic are more likely to exhibit aggression and anxiety-related behaviors at home. Veterinarians should screen cats that exhibit aggression at the veterinary clinic for behavior problems at home to institute prompt diagnosis and treatment.

Pharmacopuncture therapy for adhesive capsulitis: A pragmatic randomized controlled pilot study.

Background: Although several studies have reported the effectiveness of acupuncture treatment for adhesive capsulitis (AC), research on pharmacopuncture therapy for AC remains limited. We compared the effectiveness and safety of pharmacopuncture and physiotherapy for AC. Methods: This pragmatic, randomized, controlled, parallel-group pilot study enrolled patients with limitations of shoulder movement and a numeric rating scale (NRS) score for shoulder pain ≥5 randomized (1:1) to the pharmacopuncture therapy (PPT) and physiotherapy (PT) groups. Treatment sessions were administered twice weekly for 6 weeks, and the participants were followed up for 13 weeks after randomization. The primary outcome was the NRS score for shoulder pain, and the secondary outcomes were the visual analog scale (VAS), Shoulder Pain and Disability Index (SPADI), range of motion (ROM), patient global impression of change (PGIC), EuroQol 5-Dimension 5-Level (EQ-5D-5L), and Short Form 12 Health Survey (SF-12) scores. The intention-to-treat (ITT) analysis was set as the primary analysis. Results: Among 50 participants, for the primary endpoint (week 7) the PPT group showed a significantly superior improvement in NRS, VAS, SPADI, ROM for flexion, ROM for abduction, and EQ-5D-5L scores. The ROM for extension, ROM for adduction, physical component summary, and patient global impression of change were significantly better in the PPT than in the PT group, and these effects were sustained until week 13. Conclusion: In this pilot study, PPT showed better effects than PT, confirming the feasibility of a follow-up main study. Trial registration: Clinicaltrials.gov (NCT05292482) and cris.nih.go.kr (KCT0007198).