Fine mapping and candidate gene analysis of CaFCD1 affecting cuticle biosynthesis in Capsicum annuum L.

KEY MESSAGE: CaFCD1 gene regulates pepper cuticle biosynthesis. Pepper (Capsicum annuum L.) is an economically important vegetable crop that easily loses water after harvesting, which seriously affects the quality of its product. The cuticle is the lipid water-retaining layer on the outside of the fruit epidermis, which regulates the biological properties and reduces the rate of water-loss. However, the key genes involved in pepper fruit cuticle development are poorly understood. In this study, a pepper fruit cuticle development mutant fcd1 (fruit cuticle deficiency 1) was obtained by ethyl methanesulfonate mutagenesis. The mutant has great defects in fruit cuticle development, and the fruit water-loss rate of fcd1is significantly higher than that of the wild-type '8214' line. Genetic analysis suggested that the phenotype of the mutant fcd1 cuticle development defect was controlled by a recessive candidate gene CaFCD1 (Capsicum annuum fruit cuticle deficiency 1) on chromosome 12, which is mainly transcribed during fruit development. In fcd1, a base substitution within the CaFCD1 domain resulted in the premature termination of transcription, which affected cutin and wax biosynthesis in pepper fruit, as revealed by the GC-MS and RNA-seq analysis. Furthermore, the yeast one-hybrid and dual-luciferase reporter assays verified that the cutin synthesis protein CaCD2 was directly bound to the promoter of CaFCD1, suggesting that CaFCD1 may be a hub node in the cutin and wax biosynthetic regulatory network in pepper. This study provides a reference for candidate genes of cuticle synthesis and lays a foundation for breeding excellent pepper varieties.

Annexin A1-derived peptide Ac2-26 facilitates wound healing in diabetic mice.

Impaired wound healing is a common complication of diabetes. In diabetic wounds, macrophages present dysfunctional efferocytosis and abnormal phenotypes, which could result in excessive neutrophil accumulation and prolonged inflammation, thereby eventually hindering wound repair. ANXA1 N-terminal peptide Ac2-26 exhibits a high potential in mitigating inflammation and improving repair; however, its efficacy in diabetic wound repair remains unclear. In this study, a cutaneous excisional wound model was built in genetically diabetic mice. Ac2-26 or a vehicle solution was employed locally in wound sites. Subsequently, wound zones were measured and sampled at different time intervals post-wounding. Using hematoxylin-eosin and Masson's trichrome staining, we observed the histopathological variations and collagen deposition in wound samples. Based on immunohistochemistry and immunofluorescence, the numbers of neutrophils, macrophages, and CD206-positive macrophages in the wound samples were determined. Cytokine expression in wound samples was studied by immunoblot assay. Results showed that Ac2-26 treatment could facilitate diabetic wound closure, down-regulate the number of neutrophils, and improve angiogenesis and collagen deposition. In addition, Ac2-26 application expedited macrophage recruitment and up-regulated the percentage of macrophages expressing CD206, which is a marker for M2 macrophages. Moreover, Ac2-26 inhibited the expressions of TNF-α and IL-6 and up-regulated the expressions of IL-10, TGF-ß, and VEGFA during diabetic wound healing. Hence, based on the aforementioned findings, Ac2-26 application in diabetic wounds could exert anti-inflammatory and pro-repair effects by reducing neutrophil accumulation and facilitating M2 macrophage development.

Detection of RAGE expression and its application to diabetic wound age estimation.

With the prevalence of diabetes, it is becoming important to analyze the diabetic wound age in forensic practice. The present study investigated the time-dependent expression of receptor for advanced glycation end products (RAGE) during diabetic wound healing in mice and its applicability to wound age determination by immunohistochemistry, double immunofluorescence, and Western blotting. After an incision was created in genetically diabetic db/db mice and control mice, mice were killed at posttraumatic intervals ranging from 6 h to 14 days, followed by the sampling of wound margin. Compared with control mice, diabetic mice showed the delayed wound healing. In control and diabetic wound specimens, RAGE immunoreactivity was observed in a small number of polymorphonuclear cells (PMNs), a number of macrophages, and fibroblasts. Morphometrically, the positive ratios of RAGE in macrophages or fibroblasts considerably increased in diabetic wounds during late repair, which exceeded 60% at 7 and 10 days post-injury. There were no control wound specimens to show a ratio of >60% in macrophages or fibroblasts. By Western blotting analysis, the ratios of RAGE to GAPDH were >1.4 in all diabetic wound samples from 7 to 10 days post-injury, which were >1.8 at 10 days after injury. By comparison, no control wound specimens indicated a ratio of >1.4. In conclusion, the expression of RAGE is upregulated and temporally distributed in macrophages and fibroblasts during diabetic wound healing, which might be closely involved in prolonged inflammation and deficient healing. Moreover, RAGE is promising as a useful marker for diabetic wound age determination.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Expression of hepcidin at the choroid plexus in normal aging rats is associated with IL-6/Stat3 signaling pathway.

Accumulating evidence has revealed that brain iron concentrations increase with aging, and the choroid plexus (CP) may be at the basis of iron-mediated toxicity and the increase in inflammation and oxidative stress that occurs with aging. The mechanism involves not only hepcidin, the key hormone in iron metabolism, but also iron-related proteins and signaling-transduction molecules, such as IL-6 and signal transducer and activator of transcription 3 (Stat3). The aim of the present study was to investigate the correlation between the IL-6/Stat3 signaling pathway and hepcidin at the CP in normal aging. Quantitative real time PCR and Western blot were used to determine the alterations in specific mRNA and corresponding protein changes at the CP at ages of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months in Brown-Norway/Fischer (B-N/F) rats. The results demonstrated that hepcidin mRNA level at the CP kept stable in young rats (from 3 to 18 months), and increased with aging (from 21 to 36 months). The alterations of IL-6/p-Stat3 mRNA and protein expressions in normal aging were in accordance with that of hepcidin mRNA. Our data suggest that IL-6 may regulate hepcidin expression at the CP, upon interaction with the cognate cellular receptor, and through the Stat3 signaling transduction pathway.

Amyloid-beta transporter expression at the choroid plexus in normal aging: the possibility of reduced resistance to oxidative stress insults.

Accumulation of amyloid-beta peptides (Aß) results in amyloid burden in normal aging brain. Clearance of this peptide from the brain occurs via active transport at the interfaces separating the central nervous system (CNS) from the peripheral circulation. The present study was to investigate the change of Aß transporters expression at the choroid plexus (CP) in normal aging. Morphological modifications of CP were observed by transmission electron microscope. Real-time RT-PCR was used to measure mRNA expressions of Aß(42) and its transporters, which include low density lipoprotein receptor-related protein-1 and 2 (LRP-1 and -2), P-glycoprotein (P-gp) and the receptor for advanced glycation end-products (RAGE), at the CP epithelium in rats at ages of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33 and 36 months. At the same time, the mRNA expressions of oxidative stress-related proteins were also measured. The results showed that a striking deterioration of the CP epithelial cells and increased Aß(42) mRNA expression were observed in aged rats, and there was a decrease in the transcription of the Aß efflux transporters, LRP-1 and P-gp, no change in RAGE mRNA expression and an increase in LRP-2, the CP epithelium Aß influx transporter. Heme oxygenase-1 (HO-1) and caspase-3 expressions at the CP epithelium increased with age at the mRNA level. These results suggest the efficacy of the CP in clearing of Aß deceases in normal aging, which results in the increase of brain Aß accumulation. And excess Aß interferes with oxidative phosphorylation, leads to oxidative stress and morphological structural changes. This in turn induces further pathological cascades of toxicity, inflammation and neurodegeneration process.

Stem lodging Resistance-1 controls stem strength by positively regulating the biosynthesis of cell wall components in Capsicum annuum L.

Lodging presents a significant challenge in cultivating high-yield crops with extensive above-ground biomass, yet the molecular mechanisms underlying this phenomenon in the Solanaceae family remain largely unexplored. In this study, we identified a gene, CaSLR1 (Capsicum annuum Stem Lodging Resistance 1), which encodes a MYELOBLASTOSIS (MYB) family transcription factor, from a lodging-affected C. annuum EMS mutant. The suppression of CaSLR1 expression in pepper led to notable stem lodging, reduced thickness of the secondary cell wall, and decreased stem strength. A similar phenotype was observed in tomato with the knockdown of SlMYB61, the orthologous gene to CaSLR1. Further investigations demonstrated that CaNAC6, a gene involved in secondary cell wall (SCW) formation, is co-expressed with CaSLR1 and acts as a positive regulator of its expression, as confirmed through yeast one-hybrid, dual-luciferase reporter assays, and electrophoretic mobility shift assays. These findings elucidate the CaNAC6-CaSLR1 module that contributes to lodging resistance, emphasizing the critical role of CaSLR1 in the lodging resistance regulatory network.

Case report: Successful treatment of acute generalized pustular psoriasis of puerperium with secukinumab.

Generalized pustular psoriasis (GPP) is a rare and severe form of psoriasis presenting with erythematous, aseptic pustules. Common systemic symptoms include fever and myalgias. The presentation of GPP resembles acute generalized exanthematous pustulosis (AGEP). However, the treatment of these two pathologies differs. While AGEP is self-limiting and treated with topical corticosteroids and constrain of systemic steroids. GPP treatment avoids corticosteroid, choosing acitretin, methotrexate, and cyclosporine as first-line agents. In this case report, a 27-year-old female with a medical history of AGEP presented to the hospital with extensive erythema and pustules. Complete blood count acute phase reactant analysis revealed an elevated white blood cell count and C-reactive protein (CRP). Two histopathological examinations revealed psoriatic hyperplasia of the epidermis with keratosis, along with Kogoj and Munro micro abscesses above the spina layer. Lymphocytic and neutrophilic infiltrate was present in the superficial derma layer along with vasodilation. The patient was diagnosed with GPP according to pathological and clinical criteria. Treatment was initiated with secukinumab because of the patient's failure to respond to systemic treatment with Acitretin, methotrexate, and cyclosporin. Following 2 weeks of therapy with 300 mg of secukinumab, the pustular lesions had resolved. This study indicates the potential efficacy of secukinumab as an effective therapy that can rapidly improve the clinical symptoms of GPP.

Comprehensive Proteome and Lysine Acetylome Analysis Reveals the Widespread Involvement of Acetylation in Cold Resistance of Pepper (Capsicum annuum L.).

Pepper is a typical warmth-loving vegetable that lacks a cold acclimation mechanism and is sensitive to cold stress. Lysine acetylation plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in the resistance of pepper plants to cold stress. In this study, the proteome and acetylome of two pepper varieties with different levels of cold resistance were investigated by subjecting them to cold treatments of varying durations followed by recovery periods. In total, 6,213 proteins and 4,574 lysine acetylation sites were identified, and this resulted in the discovery of 3,008 differentially expressed proteins and 768 differentially expressed acetylated proteins. A total of 1,988 proteins were identified in both the proteome and acetylome, and the functional differences in these co-identified proteins were elucidated through GO enrichment. KEGG analysis showed that 397 identified acetylated proteins were involved in 93 different metabolic pathways. The dynamic changes in the acetylated proteins in photosynthesis and the "carbon fixation in the photosynthetic organisms" pathway in pepper under low-temperature stress were further analyzed. It was found that acetylation of the PsbO and PsbR proteins in photosystem II and the PsaN protein in photosystem I could regulate the response of pepper leaves to cold stress. The acetylation levels of key carbon assimilation enzymes, such as ribulose bisphosphate carboxylase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, and triosephosphate isomerase decreased, leading to decreases in carbon assimilation capacity and photosynthetic efficiency, reducing the cold tolerance of pepper leaves. This study is the first to identify the acetylome in pepper, and it greatly expands the catalog of lysine acetylation substrates and sites in Solanaceae crops, providing new insights for posttranslational modification studies.

Activation of α7nAChR Promotes Diabetic Wound Healing by Suppressing AGE-Induced TNF-α Production.

Diabetes frequently presents accumulation of advanced glycation end products (AGEs), which might induce excessive TNF-α production from macrophages to cause impaired wound healing. Recent studies have shown that activation of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages efficiently suppressed TNF-α synthesis. The aim of this study was to investigate the accumulation of AGEs in the wounds and determine whether PNU282987, an α7nAChR agonist, can improve wound repair by inhibiting AGE-mediated TNF-α production in a streptozotocin (STZ)-induced diabetic mouse model. Animals were assigned into four groups: wounded control group, wounded diabetic group, wounded diabetic group treated intraperitoneally with PNU282987, or wounded diabetic group treated intraperitoneally with vehicle. Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound healing that was characterized by elevated accumulation of AGEs, increased TNF-α level and macrophage infiltration, and decreased fibroblast number and collagen deposition at the late stage of repair. Besides, macrophages of diabetic wounds showed expression of α7nAChR. During late repair, PNU282987 treatment of diabetic mice significantly reduced the level of TNF-α, accelerated wound healing, and elevated fibroblast number and collagen deposition. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophage cell line, were incubated with AGEs in the presence or absence of PNU282987. TNF-α production from AGE-stimulated macrophages was significantly decreased by PNU282987 in a dose-dependent manner. Furthermore, PNU282987 significantly inhibited AGE-induced nuclear factor-κB (NF-κB) activation and receptor for AGE (RAGE) expression. These results strongly suggest that activating α7nAChR can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages.

Analysis of Forensic Autopsy in 120 Cases of Medical Disputes Among Different Levels of Institutional Settings.

Despite advances in medical science, the causes of death can sometimes only be determined by pathologists after a complete autopsy. Few studies have investigated the importance of forensic autopsy in medically disputed cases among different levels of institutional settings. Our study aimed to analyze forensic autopsy in 120 cases of medical disputes among five levels of institutional settings between 2001 and 2012 in Wenzhou, China. The results showed an overall concordance rate of 55%. Of the 39% of clinically missed diagnosis, cardiovascular pathology comprises 55.32%, while respiratory pathology accounts for the remaining 44. 68%. Factors that increase the likelihood of missed diagnoses were private clinics, community settings, and county hospitals. These results support that autopsy remains an important tool in establishing causes of death in medically disputed case, which may directly determine or exclude the fault of medical care and therefore in helping in resolving these cases.

Time-dependent expression and distribution of Egr-1 during skeletal muscle wound healing in rats.

Recent studies have shown that early growth response factor-1 (Egr-1) plays an important role in regulation of inflammation and tissue repair, but little is known about its expression after trauma to skeletal muscles. A preliminary study on time-dependent expression and distribution of Egr-1 was performed by immunohistochemistry, immunofluorescence and Western blotting during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 45 Sprague-Dawley male rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. In the uninjured controls, Egr-1 positive staining was observed in the sarcoplasm and nuclei of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells (PMNs), a number of mononuclear cells (MNCs), fibroblastic cells (FBCs) and regenerated multinucleated myotubes showed positive reaction for Egr-1 in contused zones. By morphometric analysis, an increase in Egr-1 expression was verified at inflammatory phase after contusion, which reached a peak in the regenerated phase overlapping with the fibrotic phase during skeletal muscle wound healing. The expression tendency was further confirmed by Western blotting assay. By immunofluorescent staining for co-localization, the Egr-1-positive MNCs and FBCs in wounds were identified as macrophages and myofibroblasts. The results demonstrate that the expression of Egr-1 is up-regulated and temporally distributed in certain cell types after trauma to skeletal muscles, which may be closely involved in inflammatory response, fibrotic repair and muscle regeneration during skeletal muscle wound healing.

[Zinc supplementation effects on alcoholic liver disease and the molecular mechanism].

OBJECTIVE: To examine dietary zinc supplementation could alleviate the damage of alcoholic liver disease and the relationship with the expression of hepatocyte nuclear factor 4alpha (HNF-4alpha). METHODS: 40 adult C57 BL/6 mice were randomly divided into four groups (n = 10): control, zinc, ethanol and zinc plus ethanol, which were sacrificed after fed four different diets for 6 months. Zinc sulfate was added in the drinking water of the Zinc and Zinc Plus Ethanol group and the content was 75 mg/L. Liver regeneration was assessed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA), and the expression of HNF-4alpha was determined by RT-PCR and Western blot. And as to assess the status of oxidative stress of the mice, malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. RESULTS: Compared with the control group, the expression level of HNF-4alpha decreased significantly in the ethanol group (P < 0.05), and the content of MDA increased significantly in this group, while the content of SOD declined significantly (P < 0.05). Compared with the ethanol group, the number of PCNA-positive hepatocytes increased significantly, and the expression level of HNF-4alpha also increased in the zinc plus ethanol group (P < 0.05), and the content of SOD increased in this group, while MDA decreased significantly (P < 0.05). CONCLUSION: Long term ethanol exposure can lead to oxidoreduction imbalances which can be reversed by zinc supplementation. We suppose that zinc-enhanced liver regeneration is associated with an increase in HNF-4alpha, suggesting that dietary zinc supplementation may have beneficial effects in alcoholic liver disease.

[Three-dimensional CT imaging in the treatment of children's developmental dislocation of hip].

OBJECTIVE: To investigate the value of 3-dimensional CT in the treatment of developmental dislocation of hip (DDH). METHODS: From 2003.6 to 2007.6, the femoral neck anteversion (FNA) and morphology of acetebulum in 53 patients with DDH (61 hips) were studied by three-dimensional CT imaging. Among the patients, 12 patients were male and 41 patients were female, ranging in age from 3 to 16 years (mean 5.6 years). Thirty-four patients had dislocation in left hip joint, 11 patients had dislocation in right hip joints, and 8 patients had double dislocations. The patients were treated with Pemberton or Chiari acetabuloplasty and femoral osteotomy. After operation all the cases were obeserved by 3-dimensional CT again. The femoral neck anteversions were measured and the shapes of new acetabulum were observed. RESULTS: Among 61 hips of DDH, the maximum femoral neck anteversion was 90 degrees. The minimum was 35 degrees and the average was (45.6 +/- 11.4) degrees. Among 45 normal hips, the average femoral neck anteversion was (23.5 +/- 10. 2) degrees. The acetabulum were dysplastic according with what were found in the operation. After operation the femoral neck anteversions decreased and averaged (15.6 +/- 5.8) degrees. The femoral head containment improved. CONCLUSION: The advantages of 3D CT scan includes manifestation of acetebular morphology, correct measurement of femoral neck anteversion and evaluation of operative procedure and efficacy. The 3-dimensional CT method is deserved to use widely in the treatment of developmental dislocation of hip in children.