Skin Fibroblasts Research Articles

At-Line LC-QTOF-ESI-MS/MS Fractionation of Impatiens balsamina Linn. Coupled With a Simple DPPH for Rapid Identification and Guided Isolation of Antioxidant.

Reactive oxygen species (ROS) and ultraviolet (UV) light are significant factors to impair skin disorders. Impatiens balsamina Linn. (IB), a traditional Chinese and Thai herbal medicine, has long been used to treat skin and nail diseases, potentially due to its radical-scavenging properties. However, specific antioxidant compounds in IB have not been well defined. This work aims to rapidly identify, target, and isolate antioxidant biomarkers in IB using at-line LC-ESI-QTOF-(MS/MS) coupled with a simple DPPH assay and comprehensively investigate the antioxidant activities of IB extract and isolated biomarker. Following liquid chromatography (LC), the eluent of IB extract was split into two streams (9:1 ratio). The majority was fractionated for DPPH assay in 96-well plates, whereas 10% underwent chemical identification using ESI-QTOF-MS. Antioxidants in IB were identified, targeted, and promptly isolated through transfer from analytical LC to preparative HPLC. IB and the isolated biomarkers were evaluated for antioxidant effects using various antiradical assays and in suppressing ROS induced by UV in skin cells, keratinocytes, and fibroblasts. Thirty-one chemical constituents were identified, with four tentatively identified as potent antioxidants. Kaempferol emerged as a potential antioxidant biomarker in IB, exhibiting superior antioxidant activity in various invitro assays compared with positive controls. Both IB extract and kaempferol effectively reduced UVB-induced ROS in skin cells. This study represents the first comprehensive identification of antioxidants and chemical constituents in IB, pinpointing kaempferol as a key antioxidant biomarker. Its rapid identification using at-line techniques holds promise for advancing bioactive compound discovery in herbal medicine.

Collagen Isolated from Human Adipose Tissue and Its Cellular Affinity.

The use of collagen in cell cultures promotes cell proliferation and differentiation, and it has been commercialized. In this study, we separated and purified collagen from adipose tissue discarded during liposuction and prepared collagen-coated dishes. After collagen was identified from human adipose tissue, type identification and quantification were performed using SDS-PAGE and FPLC. Collagen type I was used to coat culture dishes. Human skin fibroblasts and human adipose tissue-derived stem cells were seeded at a density of 2.5×105 cells/mL on prepared dishes at a collagen concentration of 3 mg/mL and cultured for 7 days. Cell viability was then measured and analyzed. The WST-1 assay was used to evaluate the results. The amount of collagen in 300 g of adipose tissue was 25.5 mg for type I, 41.4 mg for type III, 10.6 mg for type IV, 6.5 mg for type V, and 15 mg for type VI. The highest rates were observed for adipose stem cells cultured on human adipose tissue-derived collagen-coated dishes. In cell cultures, cell affinity was higher when cells and the substrate used were of the same origin, and affinity was stronger when the tissue of origin was the same.

Imaging mass cytometry-based characterisation of fibroblast subsets and their cellular niches in systemic sclerosis

ObjectivesTranscriptomic data demonstrated that fibroblasts are heterogeneous with functionally diverse subpopulations. Although fibroblasts are key effector cells of fibrotic diseases such as systemic sclerosis (SSc), they have not yet been...

Design and In Vitro Evaluation of Fluorescent MOF-Core CaCO₃-PEI-FA Shell Nanoparticles for Targeted Therapy of Laryngeal Cancer Cells.

Laryngeal cancer, a common malignant respiratory tumor, is primarily treated through surgery. However, challenges such as recurrence, metastasis, and drug resistance persist. In recent years, multifunctional drug delivery systems (DDS) based on nanoparticles have shown great potential in improving drug loading and release. We developed a biocompatible core-shell nanoparticle system with a zinc-based metal-organic framework (MOF) as the core, named CP1. The shell, composed of polyethyleneimine (PEI), folic acid, and calcium carbonate, forms a composite called CaCO3-PEI-FA. This system enhances biocompatibility and increases the efficacy of biomedical applications. Encapsulating CP1 within the CaCO3-PEI-FA shell allows for the targeted delivery of the anticancer drug doxorubicin (DOX) to laryngeal cancer cells (Hep-2), resulting in the CaCO3-PEI-FA@CP1@DOX system. The CaCO3-PEI-FA composite exhibits strong fluorescence with a peak around 350nm, confirming successful synthesis and demonstrating its potential as a bioimaging probe. Importantly, the nanoparticle system without DOX showed low toxicity to normal human skin fibroblasts (HSF). In vitro cytology experiments revealed a 38% inhibition rate of Hep-2 cells after 24h, highlighting the nanocomposite's significant potential in inhibiting laryngeal cancer cell proliferation and inducing apoptosis, underscoring its promise in targeted laryngeal cancer therapy.

A Transcriptional Signature of Induced Neurons Differentiates Virologically Suppressed People Living With HIV from People Without HIV.

Direct reprogramming of skin fibroblasts into neurons reveals unique gene signatures indicative of HIV infection in the context of viral suppression.

Collagen Type VII (COL7A1) as a Longevity Mediator in Caenorhabditis elegans: Anti-Aging Effects on Healthspan Extension and Skin Collagen Synthesis

Longevity genes and senescence-related signaling proteins are crucial targets in aging research, which aims to enhance the healthy period and quality of life. Identifying these target proteins remains challenging because of the need for precise categorization and validation methods. Our multifaceted approach combined bioinformatics with transcriptomic data to identify collagen as a key element associated with the lifespan of the model organism, Caenorhabditis elegans. By analyzing transcriptomic data from long-lived mutants that involved mechanisms such as antioxidation, dietary restriction, and genetic background, we identified collagen as a common longevity-associated gene. We validated these findings by confirming that collagen peptides positively affect lifespan, thereby strengthening the validity of the target. Further verification through healthspan factors in C. elegans and functional assays in skin fibroblasts provided additional evidence of the role of collagen in organismal aging. Specifically, our study revealed that collagen type VII is a significant target protein for mitigating age-related decline. By validating these findings across different aging models and cell-based studies, we present compelling evidence for the anti-aging effects of collagen type VII, highlighting its potential as a target for promoting healthy aging. This study proposes that collagen not only serves as an indicative marker of organismal longevity across various senescence-related signaling pathways, but also offers a mechanistic understanding of skin degeneration. Consequently, collagen is an effective target for interventions aimed at mitigating skin aging. This study underscores the potential of collagen type VII (bonding collagen T7) as a therapeutic target for enhancing skin health and overall longevity.

Construction of induced pluripotent stem cell line (CSBZZUi002-A) from the fibroblast cells of a healthy female

We recruited a healthy 44-year-old female and obtained her skin fibroblasts. Subsequently, the induced pluripotent stem cell line was successfully established using non-integrated reprogramming technology. The cell line had a normal karyotype and has been confirmed to have good pluripotency through the detection of pluripotency markers and detection of teratoma formation. This cell line can serve as an effective control for studying the cellular pathological mechanisms of other specific mutations.

Wound Healing, Metabolite Profiling, and In Silico Studies of Aspergillus terreus.

Burn injuries, which significantly affect global public health, require effective treatment strategies tailored to varying severity. Fungi are considered a sustainable, easily propagated source for lead therapeutic discovery. In this study, we explored the burn wound healing potential of Aspergillus terreus through a combination of in vitro, in vivo, metabolite profiling, and in silico analysis. The in vitro scratch assays performed with human skin fibroblast cells showed promising wound healing activity. Furthermore, the burn-induced rats model showed a marked improvement in cutaneous wound healing, evidenced by an accelerated rate of wound closure and better skin regeneration after A. terreus extract treatment at 14 days. The results of this study demonstrated significant enhancements in wound closure and tissue regeneration in the treated rat model, surpassing the outcomes of standard treatments. This controlled healing process, evidenced by superior collagen synthesis and angiogenesis and confirmed by histopathological studies, suggests that A. terreus has potential beyond the traditionally studied fungal metabolites. The metabolite profiling of 27 bioactive compounds was further investigated by docking analysis for the potential inhibition of the NF-κB pathway, which has an important function in inflammation and wound repair. The compounds eurobenzophenone A (7), aspernolide D (16), asperphenalenone A (23), aspergilate D (15), kodaistatin A (18), and versicolactone A (14) showed the highest binding affinity to the target protein with a pose score of -16.86, -14.65, -12.65, -12.45, -12.19, and -12.08 kcal/mol, respectively. Drug-likeness properties were also conducted. The findings suggest the potential wound healing properties of A. terreus as a source for lead therapeutic candidate discovery.

Unlocking the wound healing potential of Anastatica hierochuntica L. (Kaff Maryam) Extract's nanosuspension: UPLC-MS/MS metabolic profiling, formulation development and statistical optimization

BackgroundAnastatica hierochuntica L. (Kaff Maryam) is a desert plant with documented therapeutic applications. Various medicinal uses have been reported for the plant extract such as antioxidant, anti-inflammatory, antifungal, antimicrobial, hypoglycemic, hypolipidemic, and hepatoprotective activities. The abundant plants constituents are promising and encourage the investigation of novel therapeutic uses. The principal objective of this study was to identify the bioactive constituents of A. hierochuntica (AH) by phytochemical profiling of its ethanolic extract, alongside an investigation of its wound healing efficacy after being formulated into nanosuspension for topical localized wound treatment. MethodA comprehensive metabolomics study of the ethanolic extract of A. hierochuntica aerial parts was assessed using UPLC MS/MS technique. Additionally, the preparation of AH nanosuspension was executed utilizing the antisolvent-precipitation method. A D-optimal experimental design was adopted to investigate the influence of variables such as: stabilizer ratio, antisolvent: solvent ratio, and stabilizer type on particle size (PS), polydispersity index (PDI), and zeta potential (ZP). The design also was employed in the optimization process. The selected optimal formula was further assessed for size characteristics, morphological identification by TEM, storage stability, FTIR, cytocompatibility with human skin fibroblast (HSF), and scratch assay on HSF to assess wound healing aptitude. ResultsChemical profiling of the aerial parts revealed an abundance of glucosinolates, flavonoids, flavolignans, and fatty acids. The optimized AH-nanosuspension showed small particle size (128.87 ± 26 nm), small PDI (0.136 ± 0.05), and high ZP absolute value (−37.53 ± 0.52 mV). Furthermore, TEM photography of the optimal nanosuspension formula exhibited circular non-aggregating nanosized particles. FTIR confirmed the successful formulation of the AH ethanolic extract into nanosuspension with no physicochemical interactions. Additionally, AH-nanosuspension demonstrated cytocompatibility with HSF with IC50 = 58.317 μg/mL. Moreover, both AH pure extract and AH-nanosuspension exhibited a remarkable cell migration rate and wound closure % which propounds an auspicious wound healing potential. ConclusionThe study accentuates the supremacy of the nanosized delivery approach of AH aerial parts ethanolic extract and warrants further in-vivo investigations to establish its clinical utility for wound management.

Silencing of ERRα gene represses cell proliferation and induces apoptosis in human skin fibroblasts.

Estrogen‑related receptor (ERR) is an orphan nuclear receptor structurally akin to the estrogen receptor. ERR is expressed in tissues with active energy metabolism and regulates intracellular metabolic functions. Additionally, ERRs are known to be strongly expressed in the epidermis of skin tissue, but their functions are unknown. The present study investigated the function of ERRα in human skin fibroblasts. ERRα expressed in human dermal fibroblast TIG113 was knocked down using small interfering (si)RNA and gene expression was comprehensively analyzed using microarrays 48 h later. Pathway analysis was performed using Wikipathways on genes exhibiting expression changes of ≥1.5‑fold. Expression of cell cycle‑related and apoptosis‑related genes was compared using reverse transcription‑quantitative PCR. After treating TIG113 cells with siERRα for 72 h, cell proliferation was assessed using the Cell Counting Kit‑8 or a scratch wound healing assay and apoptotic cells were measured using the Poly Caspase Assay Kit. Cell cycle analysis was performed using flow cytometry. The expression of the ERRα gene was suppressed by siRNA. The expression of genes associated with cell cycle‑related pathways were decreased while that of those associated with apoptosis‑related pathways increased. Furthermore, the expression of cell cycle‑related genes such as cell division cycle 25C, cyclin E and cyclin B1 was decreased and the expression of apoptosis‑related genes such as caspase3 and Fas cell surface death receptor was increased. Cell proliferation was suppressed and the number of apoptotic cells increased ~2‑fold in ERRα‑knockdown TIG113 cells. Cell cycle analysis revealed that the number of cells in the Sub‑G1 phase increased and that in the S and G2/M phases decreased. The present study suggested that ERRα is an essential for the survival of human skin fibroblasts.

Inhibiting Triggering Receptor Expressed on Myeloid Cells-1 signaling to ameliorate skin fibrosis.

Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. Transforming growth factor-beta (TGF-β) is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used novel ligand-independent TREM-1 inhibitors in order to investigate the pathogenic role of TREM-1 in SSc, using preclinical models of fibrosis, and explanted SSc skin fibroblasts. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.

Streptomyces fradiae Mitigates the Impact of Potato Virus Y by Inducing Systemic Resistance in Two Egyptian Potato (Solanum tuberosum L.) Cultivars

In this study, the impact of culture media filtrate of QD3 actinobacterial isolate on two potato cultivars, Spunta and Diamond, infected with potato virus Y (PVY) was investigated. Various parameters, including infection percentage, PVY virus infectivity, disease severity scoring, PVY optical density, photosynthetic and defense-related biochemical markers, enzymatic profiling, phenolic compounds, proline content, salicylic acid levels, and growth and yield parameters, were assessed to elucidate the potential of the QD3 actinobacterial isolate culture filtrate in mitigating PVY-induced damage. The physiological and biochemical characteristics of the QD3 actinobacterial isolate, including its salinity tolerance, pH preferences, and metabolic traits, were investigated. Molecular identification via 16S rRNA gene sequencing confirmed its classification as Streptomyces fradiae QD3, and it was deposited in GenBank with the gene accession number MN160630. Distinct responses between Spunta and Diamond cultivars, with Spunta displaying greater resistance to PVY infection. Notably, pre-infection foliar application of the QD3 filtrate significantly reduced disease symptoms and virus infection in both cultivars. For post-PVY infection, the QD3 filtrate effectively mitigated disease severity and the PVY optical density. Furthermore, the QD3 filtrate positively influenced photosynthetic pigments, enzymatic antioxidant activities, and key biochemical components associated with plant defense mechanisms. Gas chromatography‒mass spectrometry (GC‒MS) analysis revealed palmitic acid (hexadecanoic acid, methyl ester) and oleic acid (9-octadecanoic acid, methyl ester) as the most prominent compounds, with retention times of 23.23 min and 26.41 min, representing 53.27% and 23.25%, respectively, of the total peak area as primary unsaturated fatty acids and demonstrating antiviral effects against plant viruses. Cytotoxicity assays on normal human skin fibroblasts (HSFs) revealed the safety of QD3 metabolites, with low discernible toxicity at high concentrations, reinforcing their potential as safe and effective interventions. The phytotoxicity results indicate that all the seeds presented high germination rates of approximately 95–98%, suggesting that the treatment conditions had no phytotoxic effect on the Brassica oleracea (broccoli) seeds, Lactuca sativa (lettuce) seeds, and Eruca sativa (arugula or rocket) seeds. Overall, the results of this study suggest that the S. fradiae filtrate has promising anti-PVY properties, influencing various physiological, biochemical, and molecular aspects in potato cultivars. These findings provide valuable insights into potential strategies for managing PVY infections in potato crops, emphasizing the importance of Streptomyces-derived interventions in enhancing plant health and crop protection.

The role of DNA methylation in chondrogenesis of human iPSCs as a stable marker of cartilage quality

BackgroundLack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act.MethodsWe applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC).ResultsMethylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation.ConclusionsWe did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice.

Early Effects of Porcine Placental Extracts and Stem Cell-Derived Exosomes on Aging Stress in Skin Cells.

The initial efficacy of placental extracts (Pla-Exts) and human mesenchymal stem-cell-derived exosomes (hMSC-Exo) against aging-induced stress in human dermal fibroblasts (HDFs) was examined. The effect of Pla-Ext alone, hMSC-Exo alone, the combined effect of Pla-Ext and hMSC-Exo, and the effect of hMSC-Exo (Pla/MSC-Exo) recovered from cultures with Pla-Ext added to hMSC were verified using collagen, elastin, and hyaluronic acid synthase mRNA levels for each effect. Cells were subjected to photoaging (UV radiation), glycation (glycation end-product stimulation), and oxidation (H2O2 stimulation) as HDF stressors. Pla-Ext did not significantly affect normal skin fibroblasts with respect to intracellular parameters; however, a pro-proliferative effect was observed. Pla-Ext induced resistance to several stresses in skin fibroblasts (UV irradiation, glycation stimulation, H2O2 stimulation) and inhibited reactive oxygen species accumulation following H2O2 stimulation. Although the effects of hMSC-Exo alone or the combination of hMSC-Exo and Pla-Ext are unknown, pretreated hMSC-Exo stimulated with Pla-Ext showed changes that conferred resistance to aging stress. This suggests that Pla-Ext supplementation may cause some changes in the surface molecules or hMSC-Exo content (e.g., microRNA). In skin cells, the direct action of Pla-Ext and exosomes secreted from cultured hMSCs pretreated with Pla-Ext (Pla/MSC-Exo) also conferred resistance to early aging stress.

Sall4 and Gata4 induce cardiac fibroblast transition towards a partially multipotent state with cardiogenic potential

Cardiac cellular fate transition holds remarkable promise for the treatment of ischemic heart disease. We report that overexpressing two transcription factors, Sall4 and Gata4, which play distinct and overlapping roles in both pluripotent stem cell reprogramming and embryonic heart development, induces a fraction of stem-like cells in rodent cardiac fibroblasts that exhibit unlimited ex vivo expandability with clonogenicity. Transcriptomic and phenotypic analyses reveal that around 32 ± 6.4% of the expanding cells express Nkx2.5, while 13 ± 3.6% express Oct4. Activated signaling pathways like PI3K/Akt, Hippo, Wnt, and multiple epigenetic modification enzymes are also detected. Under suitable conditions, these cells demonstrate a high susceptibility to differentiating into cardiomyocyte, endothelial cell, and extracardiac neuron-like cells. The presence of partially pluripotent-like cells is characterized by alkaline phosphatase staining, germ layer marker expression, and tumor formation in injected mice (n = 5). Additionally, significant stem-like fate transitions and cardiogenic abilities are induced in human cardiac fibroblasts, but not in rat or human skin fibroblasts. Molecularly, we identify that SALL4 and GATA4 physically interact and synergistically stimulate the promoters of pluripotency genes but repress fibrogenic gene, which correlates with a primitive transition process. Together, this study uncovers a new cardiac regenerative mechanism that could potentially advance therapeutic endeavors and tissue engineering.

PTB Regulates Keloid Fibroblast Migration and Proliferation Through Autophagy.

Keloid disease is a chronic fibroproliferative disease that occurs after tissue injury, and the currently available treatments are unsatisfactory. We aimed to explore the level of autophagy in keloid fibroblasts (KFbs) and adjacent normal fibroblasts (NFbs). In addition, whether polypyrimidine tract-binding protein (PTB) regulates the biological functions of KFbs via autophagy was also investigated. The morphology of fibroblasts in normal skin and keloids was observed transmission electron microscopy. We silenced PTB with PTB-specific siRNA to determine whether PTB-regulated KFb proliferation. Acridine orange and LysoTracker Red staining was performed to label acidic compartments. Interestingly, when autophagy was inhibited by wortmannin, the PTB knockdown-mediated decrease in KFb migration and proliferation was abolished, while the collagen I and III levels were not altered; these results indicated that PTB regulated the migration and proliferation of KFbs via autophagy, while collagen synthesis occurred independently of PTB regulation. Many activities related to the survival and function of KFbs are controlled by PTB. Transmission electron microscopy revealed more autophagosomes and autolysosomes in KFbs than in NFbs. PTB induced autophagy in KFbs, as demonstrated by the significantly greater number of autophagosomes in KFbs after PTB knockdown, which was revealed by acridine orange and LysoTracker staining. Our study is the first to show that PTB regulates the migration and proliferation of KFbs via autophagy and that PTB regulates collagen synthesis in KFbs in an autophagy-independent manner. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Establishment of the first dermal fibroblast cell line derived from the Indo-Pacific Bottlenose Dolphin (Tursiops aduncus) and its response to pollutant exposure

The Indo-Pacific bottlenose dolphin (Tursiops aduncus), classified as a new species since 1998, is underexplored in ecotoxicology owing to ethical constraints and the lack of specific in vitro models. Herein, we established the first skin fibroblast cell line (TaSF) from an Indo-Pacific bottlenose dolphin stranded along the Pearl River Estuary, China. TaSF cells exhibited strong proliferation in early passages but ceased mitosis at passage 19, likely owing to reaching the Hayflick limit. Morphology and immunofluorescence tests confirmed the fibroblastic nature of these cells. Karyotyping revealed 21 pairs of autosomes and 1 pair of sex chromosomes (XY), consistent with many cetaceans. To facilitate long-term future studies, TaSF cells were immortalized with exogenous simian virus 40 T antigen, creating the TaSFT cell line. The cytotoxic effects of 27 contaminants, including 6 organotins (OTs), 10 per- and polyfluoroalkyl substances (PFASs), and 11 phthalates (PAEs), on TaSFT cells were evaluated after 24-h exposure. Among these chemicals, OTs exhibited the strongest cytotoxicity, and both OTs and PFASs showed structure-related toxicity. These findings confirm the feasibility of TaSFT cells as a novel tool for ecotoxicity research on the Indo-Pacific bottlenose dolphin and highlight the need for further investigation into the environmental contaminant pressures on this species.

Clinical characteristics and genetics functional analysis of two children with Spinal muscular atrophy

To explore the characteristics of SMN1 gene variants and carry out functional verification for two children with Spinal muscular atrophy (SMA). Two male children with complicated SMA diagnosed at the Children's Hospital Affiliated to Capital Institute of Pediatrics respectively in July 2021 and April 2022 due to delayed or retrograde motor development were selected as the study subjects. Clinical data of the children were collected. Primary culture of skin fibroblasts was carried out, and peripheral blood samples were collected from both children and their parents. Multiplex ligation-dependent probe amplification, combined long-range PCR and nested PCR, and Sanger sequencing were carried out to detect the copy number and variants of the SMN1 gene. Absolute quantitative real-time PCR, Western blotting and immunofluorescence were used to determine the transcriptional level of the SMN gene, expression of the SMN protein, and the number of functional SMN protein complexes (gems body), respectively. This study was approved by the Children's Hospital Affiliated to Capital Institute of Pediatrics (Ethics No. SHERLLM2021009). Child 1, a 1-year-old boy, was clinically diagnosed with type 1 SMA. Child 2, a 2-and-a-half-year-old boy, was clinically diagnosed with type 3 SMA. Both children were found to harbor a paternally derived SMN1 deletion and a maternally derived SMN1 gene variant, namely c.824G>T (p.Gly275Val) and c.884A>T (p.*295Leu). Compared with the normal controls and carriers, the levels of full-length SMN1 transcripts in their peripheral blood and skin fibroblast cell lines were significantly decreased (P < 0.05), and the levels of SMN protein normalized to that of β-actin, and the numbers of gems bodies in the primary fibroblast cells were also significantly lower (P < 0.05). Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic (PS3+PM3+PM5+PP3; PS3+PM3+PM4+PP3). Following the diagnosis, both children had received nusinersen treatment. Although their motor function was improved, child 1 still died at the age of 2 due to severe pulmonary infection. The walking ability of child 2 was significantly improved, and his prognosis appeared to be good. Two cases of clinically complicated SMA have been confirmed by genetic testing and experimental studies, which has provided a reference for their accurate treatment.

Tyrosinase Inhibitory Activity of Crude Procyanidin Extract from Green Soybean Seed and the Stability of Bioactive Compounds in an Anti-Aging Skin Care Formulation

Green soybean (Glycine max L.) seed contains a high procyanidin content and high antioxidant activity. Moreover, ultrasound-assisted extraction (UAE) has proved to be advantageous in providing high extraction efficiency. Hence, this study aimed to extract procyanidins from green soybean seeds (GSSs) using UAE. This study also evaluated the inhibitory activities of tyrosinase and the cytotoxic effects of crude procyanidin extract. The extract exhibited maximum levels of bioactive components and antioxidant capacity when subjected to a temperature of 15 °C and an extraction time of 20 min. The crude procyanidin extract at a concentration of 10 mg/mL inhibited the tyrosinase enzyme by more than 60%, and the half-maximal inhibitory concentration (IC50) value obtained for the extract was 6.85 ± 0.81 mg/mL. This result was much greater than the IC50 value obtained for kojic acid (0.089 ± 0.08 mg/mL), which was used as a positive control. For the cytotoxicity assessment, the results indicated that the crude procyanidin extract showed no cytotoxicity and actually stimulated the growth of human skin fibroblast cells. More than 80% of the bioactive compounds (total phenolic content (TPC), total flavonoid content (TFC), procyanidin content (PC)) and antioxidant activities (DPPH and FRAP) of the crude extract powder were retained at 38.68 ± 0.01 mg GAE/g, 16.07 ± 0.01 mg CAE/g, 9.24 ± 0.01 mg PC/g, 359.8 ± 0.72 μM Trolox eq/g, and 1640 ± 2.86 μM Trolox eq/g, respectively, after 12 weeks of storage at 25 °C. The crude procyanidin extract powder was then included in a facial serum formulation and tested for pH value and physical evaluation. The stability of the crude procyanidin extract facial serum was shown to be greater for bioactive compounds and antioxidant activity when stored at a temperature of 4 °C than when stored at a temperature of 25 °C. These results suggest that the GSS extracts obtained via ultrasonication show promise for use in cosmeceutical formulations for whitening skincare products.

Investigating Concomitant RAG-2 and LRBA Mutations in SCID and Autoimmunity.

Inborn errors of immunity (IEI) are a large heterogenous group of diseases characterized by immunodeficiency, immune dysregulation, allergy, auto-inflammation and predisposition for malignancies. Most are inherited in an autosomal recessive trait. We studied a patient with severe combined immunodeficiency (SCID) and immune dysregulation who harbored two distinct bi-allelic IEI-associated genetic mutations. Clinical, immunological and genetic data were collected. Genetic investigation included whole exome sequencing on DNA extracted from skin fibroblasts. Family segregation was performed by Sanger sequencing. Immunological evaluation included absolute and functional evaluation of lymphocytes and chimerism analysis post hematopoietic stem cell transplantation (HSCT). Treg subsets, LRBA and CTLA4 expression levels were measured by flow-cytometric analysis. A nineteen-year-old female patient from a consanguine background underwent unconditioned matched sibling related HSCT during infancy due to clinical presentation of SCID with an Omenn phenotype. At that time her underlying genetic defect was not defined. Years after HSCT, severe auto-immune phenomena were noted, including a systemic lupus erythematosus-like syndrome and ophthalmic manifestations. Genetic evaluation revealed bi-allelic homozygous mutations in RAG-2 (c.685C>T, p.Arg229Trp) and a previously undescribed mutation in LRBA (c.3325G>T, p.Asp1109Tyr). LRBA and CTLA4 expression levels were normal, suggesting that the LRBA variant identified in these kindred is unlikely to be pathogenic. Multiple genetic defects causing complex IEIs may be identified in the same individual in highly consanguineous populations. Functional immunological testing is essential for evaluation of novel genetic variants.