Specific Markers Research Articles

Microfluidic Fabrication of Gelatin-Nano Hydroxyapatite Scaffolds for Enhanced Control of Pore Size Distribution and Osteogenic Differentiation of Dental Pulp Stem Cells.

The combination of gelatin and hydroxyapatite (HA)has emerged as a promising strategy in dental tissue engineering due to its favorable biocompatibility, mechanical properties, and ability to support cellular activities essential for tissue regeneration, rendering them ideal components for hard tissue applications. Besides, precise control over interconnecting porosity is of paramount importance for tissue engineering materials. Conventional methods for creating porous scaffolds frequently encounter difficulties in regulating pore size distribution. This study demonstrates the fabrication of gelatin-nano HA scaffolds with uniform porosity using a T-type junction microfluidic device in a single-step process. Significant improvements in control over the pore size distribution are achieved by regulating the flow parameters, resulting in effective and time-efficient manufacturing comparable in quality to the innovative 3D bioprinting techniques. The overall porosity of the scaffolds exceeded 60%, with a remarkably narrow size distribution. The incorporation of nano-HAinto 3D porous gelatin scaffolds successfully induced osteogenic differentiation in stem cells at both the protein and gene levels, as evidenced by the significant increase in osteocalcin (OCN), an important marker of osteogenic differentiation. The OCN levels are 26 and 43 times higher for gelatin and gelatin-HA scaffolds, respectively, compared to the control group.

Identification of Cervi cornu pantotrichum and its mixed varieties by UHPLC-QTOF-MS and UHPLC-TQS-MS/MS

Sika deer (Cervus nippon Temminck) and red deer (Cervus elaphus Linnaeus) are the two creatural sources of velvet antlers, which are recorded in the Chinese Pharmacopoeia (2020 edition). However, the counterfeits-moose (Alces alces Linnaeus) antlers are selling as genuine products in the market. In order to strengthen antler quality control and market supervision, a UHPLC-QTOF-MS coupled with chemometrics analysis and UHPLC-TQS-MS were developed and applied to distinguish the Velvet Antler of moose from that of sika deer and red deer. Firstly, the QTOF-MS quantized data obtained from samples treated with trypsin were subjected to principal component analysis (PCA) to classify these three kinds of antlers. Then, the potential differential marker peptides were found with orthogonal partial least-squares discriminant analysis (OPLS-DA). Finally, validation of these differential marker peptides was assessed using UHPLC-QTOF-MS, and the results showed that the specific ion pairs (m/z 724.8 → 518.1, m/z 724.8 → 938.3, m/z 732.8 → 526.3, m/z 732.8 → 784.3) were detected in moose deer antler, not in velvet antler of sika deer (Cervus nippon Temminck) and red deer (Cervus elaphus Linnaeus). The methods in the paper and specific ion pairs could be successfully applied to distinguish moose antlers from sika deer antlers and red deer antlers.

TTF-1 is a highly sensitive but not fully specific marker for pulmonary and thyroidal cancer: a tissue microarray study evaluating more than 17,000 tumors from 152 different tumor entities.

Thyroid transcription factor 1 (TTF-1) immunohistochemistry (IHC) is routinely used for the distinction of primary pulmonary adenocarcinomas. However, TTF-1 can also occur in other malignancies. A tissue microarray containing 17,772 samples from 152 different tumor types was analyzed. Napsin-A, CK20, SATB2, FABP1, and Villin-1 IHC data were available from previous studies. TTF-1 staining was seen in 82 of 152 tumor categories including thyroidal cancers (19-100%), adenocarcinomas (94%), neuroendocrine tumors (67%) of the lung, small cell neuroendocrine carcinomas (71-80%), mesenchymal tumors (up to 42%), and thymomas (39%). Comparative analysis of TTF-1 and Napsin-A revealed a sensitivity/specificity of 94%/86% (TTF-1), 87%/98% (Napsin-A), and 85%/99.1% (TTF-1 and Napsin-A) for the distinction of pulmonary adenocarcinomas. Combined analysis of TTF-1 and enteric markers revealed a positivity for TTF-1 and at least one enteric marker in 22% of pulmonary adenocarcinomas but also a TTF-1 positivity in 6% of colorectal, 2% of pancreatic, and 3% of gastric adenocarcinomas. TTF-1 is a marker of high sensitivity but insufficient specificity for pulmonary adenocarcinomas. A small fraction of TTF-1-positive gastrointestinal adenocarcinomas represents a pitfall mimicking enteric-type pulmonary adenocarcinoma. Combined analysis of TTF-1 and Napsin-A improves the specificity of pulmonary adenocarcinoma diagnosis.

12331 Overcoming Limitations Of Identifying Proliferating β-cells In Dispersed Primary Islets Culture For Drug Discovery

Abstract Disclosure: H. Xu: None. S. Rebecca: None. S. Lee: None. J.P. Annes: None. Tremendous efforts have been made with screening platforms to identify islet β-cell mitogens that may be used to treat diabetes. However, promising compounds induce profound proliferation of islet non-β-cells in available screening platforms, calling for both β-cell targeted drug discovery and rigorous identification of β-cell identity to reduce false positivity. First, we used an established drug discovery dispersed islet cell-based platform(2D) to test the replication-promoting activity of GNF2133, a selective DYRK1A inhibitor that promotes β-cell proliferation, in isolated islets from human, rat and mouse. After 72 h in vitro culture, replication of non-β cells (PDX/Insulin negative) contributed to over 60% of the total replicating cells (Ki67+) in all species (human: 64%, rat: 71%, mouse: 67%). Mouse islets, which exhibit the greatest non-β-cell replication response upon GNF2133 treatment (4-fold above baseline, [50 nM]), were selected for subsequent studies. Next, we evaluated identification efficiency of β cell markers in this image-based platform. β cells, selected by the quantification value of insulin intensity, comprised 60-70% of total cell population at 48 h, 50-60% at 72 h and 20-30% at 96 h (p< 0.0001 for both comparisons: 48 h vs 72 h, 72 h vs 96 h). Basal replication of β-cells (Insulin+Ki67+) was 2.80%, 2.32%, 0.85% at 48 h, 72 h and 96 h, respectively. By comparison, the proportion of non-β cells showed an increasing trend as 2.48%, 6.49%, 8.68%, correspondingly. Identification with PDX1, another β cell marker, exhibited the same findings. Prolonged culture of dispersed islet cells, as these results indicate, favors growth of non-β-cells over β-cells. In addition, proliferating β-cells express lower levels of differentiation markers makes their discernment from replicating non-β cells more challenging. 窗体顶端 To minimize the duration of monolayer culture, replication induction was measured using intact islets with addition of BrdU during the last 24 h of culture. Then, islets were either directly fixed (3D) or dispersed and cultured for an additional 36 hour before being fixed (3D+2D). Immunostaining showed 70-80% PDX1 positive cells from both methods, which resembles in vivo β cell distribution within primary mouse islets. Basal β-cell replication in 3D+2D method was 0.30%, similar to in vivo BrdU labelling level. Upon mitogen treatment, PDX1+BrdU+ cells increased to 6.57% in 3D+2D method, slightly lower than 10.02% in 3D assay (p<0.05) yet significantly higher than 1.15% in 2D assay (<0.01). In summary, we developed a 3D+2D assay that enhances β-cell survival and the β-cell replication-induction response. This platform, while offering reduced throughput, provides a robust platform for discovery of β-cell replication-promoting compounds. Further validation with human islets is needed. Presentation: 6/2/2024

4-aminopyridine attenuates inflammation and apoptosis and increases angiogenesis to promote skin regeneration following a burn injury in mice

Severe thermal skin burns are complicated by inflammation and apoptosis, which delays wound healing and contributes to significant morbidity. Diverse treatments demonstrate limited success in mitigating these processes to accelerate healing. Agents that alter cell behavior to improve healing would alter treatment paradigms. We repurposed 4-aminopyridine (4-AP), a drug approved by the US FDA for multiple sclerosis, to treat severe burns in mice (10-week-old C57BL/6 J male mice weighing 25 ± 3 g). We found that 4-AP, in the early stages of burn healing, significantly reduced the expression of pro-inflammatory cytokines IL1β and TNFα while increasing the expression of anti-inflammatory markers CD206, ARG-1, and IL10. We demonstrated increased intracellular calcium effects of 4-AP through Orai1-pSTAT6 signaling, where 4-AP significantly mitigated inflammatory effects by promoting M2 macrophage differentiation in in-vitro macrophages and post-skin burn tissues. 4-AP attenuated apoptosis, with decreases in apoptotic markers BAX, caspase-9, and caspase-3 and increases in anti-apoptotic markers BCL2 and BCL-XL. Furthermore, 4-AP promoted angiogenesis through increases in the expression of CD31, VEGF, and eNOS. Together, these likely contributed to accelerated burn wound closure, as demonstrated in increased keratinocyte proliferation (K14) and differentiation (K10) markers. In the later stages of burn healing, 4-AP increased TGFβ and FGF levels, which are known to mark the transformation of fibroblasts to myofibroblasts. This was further demonstrated by an increased expression of α-SMA and vimentin, as well as higher levels of collagen I and III, MMP 3, and 9 in mice treated with 4-AP. Our findings support the idea that 4-AP may have a novel, clinically relevant therapeutic use in promoting burn wound healing.

Compound K Promotes Megakaryocytic Differentiation by NLRP3 Inflammasome Activation.

Platelets are essential blood components that maintain hemostasis, prevent excessive bleeding, and facilitate wound healing. Reduced platelet counts are implicated in various diseases, including leukemia, hepatitis, cancer, and Alzheimer's disease. Enhancing megakaryocytic differentiation is a promising strategy to increase platelet production. Compound K (CK), a major bioactive metabolite of ginsenosides from Panax ginseng, has demonstrated anti-cancer and neuroprotective properties. In this study, we investigated the effects of CK on megakaryocytic differentiation and apoptosis in chronic myeloid leukemia (CML) cell lines K562 and Meg-01. CK treatment significantly upregulated the mRNA expression of key megakaryocytic differentiation markers, including CD61, CD41, and CD42a, and promoted the formation of large, multinucleated cells in K562 cells. Additionally, flow cytometry analysis revealed that CK at 5 µM induced apoptosis, a critical process in thrombocytopoiesis, in both K562 and Meg-01 cells. RT2 Profiler PCR array analysis further identified a marked increase in the expression of genes associated with the activation of the NLRP3 inflammasome in CK-treated K562 and Meg-01 cells. This study is the first to demonstrate that CK promotes megakaryocytic differentiation and apoptosis through the activation of the ERK/EGR1 and NLRP3 inflammasome pathways. These findings suggest that CK may enhance platelet production, indicating its potential as a therapeutic candidate for platelet-related disorders and other associated diseases.

Chemical profiling and comparative analysis of different parts of Asarum heterotropoides using SPME-GC-QTOF-MS and LC- Orbitrap -MS

Asari radix et rhizoma is the sole plant from the Aristolochiaceae family officially sanctioned for medicinal in China, primarily employed for treating colds and headaches, and is widely utilized in clinical practice. Initially, the entire plant was specified for medicinal use, but since 2005, the authorized part has been restricted to the roots and rhizomes. The chemical constituents are directly linked to its efficacy and safety, yet a comparative analysis of the chemical profiles between the overground and underground parts has not been reported. This paper represents the first comparative study of the chemical constituents in the two parts, achieved through the synergistic application of solid phase micro extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS) and liquid chromatography Orbitrap MS (LC-Orbitrap-MS). Using SPME-GC-MS, 51 constituents were identified from both parts, with 89 % being shared components, indicating a close similarity in their volatile compositions. Through LC-Orbitrap-MS, 308 constituents were identified, sharing 76 % commonality, revealing a more pronounced disparity in non-volatile components. Plant metabolomics screening pinpointed 8 volatile and 14 non-volatile components capable of distinguishing the two parts, with the latter being more stable and thus better suited as markers for differentiation. This research furnishes a scientific rationale for selecting distinct parts of Asari radix et rhizoma and for implementing monitoring strategies in clinical application.

Effect of recombinant irisin on recombinant human bone morphogenetic protein-2 induced osteogenesis and osteoblast differentiation

Osteoporotic fragility fractures substantially impact aging societies, necessitating long-term care and increasing healthcare costs. Myokine irisin, secreted by skeletal muscle, influences bone metabolism; however, a comprehensive understanding of the mechanisms by which irisin affects bone metabolism is still lacking. Therefore, this study aimed to explore the effects of irisin on osteogenesis and osteoblast differentiation triggered by bone morphogenetic protein-2 (BMP-2). We used 4-week-old male ICR mice and implanted polyethylene glycol pellets containing recombinant human BMP-2 (rh-BMP-2) into the left dorsal muscle pouch. Mice received weekly intraperitoneal injections of either phosphate-buffered saline or recombinant irisin (re-irisin). Ectopic bone formation was evaluated 3 weeks post-surgery using micro-computed tomography (μ-CT) and histological analysis. In vitro experiments, C2C12 cells were treated with or without rh-BMP-2 and re-irisin, and we assessed osteoblast differentiation markers, e.g., runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and osteopontin, using real-time reverse transcription-polymerase chain reaction. The μ-CT analyses showed that re-irisin significantly increased bone mineral content and bone volume of ectopic bones newly formed by rh-BMP-2. The gene expressions of the osteoblast markers were significantly increased by rh-BMP-2 and further upregulated by re-irisin. The treatment of cyclic AMP response element-binding protein (CREB) small interfering RNA attenuated these effects, suggesting that CREB signaling pathway was involved in rh-BMP-2/re-irisin-induced osteoblastic differentiation. This study demonstrates the potential of irisin to enhance osteogenesis through BMP signaling, offering insights for osteoporosis treatment and highlighting irisin as a promising therapeutic target for improving bone health and extending a healthy lifespan.

Mucosa-like differentiation of head and neck cancer cells is inducible and drives the epigenetic loss of cell malignancy

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue, distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection.

Sustained BMP-2 delivery via alginate microbeads and polydopamine-coated 3D-Printed PCL/β-TCP scaffold enhances bone regeneration in long bone segmental defects

Background/ObjectiveRepair of long bone defects remains a major challenge in clinical practice, necessitating the use of bone grafts, growth factors, and mechanical stability. Hence, a combination therapy involving a 3D-printed polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) scaffold coated with polydopamine (PDA) and alginate microbeads (AM) for sustained delivery of bone morphogenetic protein-2 (BMP-2) was investigated to treat long bone segmental defects. MethodsSeveral in vitro analyses were performed to evaluate the scaffold osteogenic effects in vitro such as PDA surface modification, namely, hydrophilicity and cell adhesion; cytotoxicity and BMP-2 release kinetics using CCK-8 assay and ELISA, respectively; osteogenic differentiation in canine adipose-derived mesenchymal stem cells (Ad-MSCs); formation of mineralized nodules using ALP staining and ARS staining; and mRNA expression of osteogenic differentiation markers using RT-qPCR. Bone regeneration in femoral bone defects was evaluated in vivo using a rabbit femoral segmental bone defect model by performing radiography, micro-computed tomography, and histological observation (hematoxylin and eosin and Masson's trichrome staining). ResultsThe PDA-coated 3D-printed scaffold demonstrated increased hydrophilicity, cell adhesion, and cell proliferation compared with that of the control. BMP-2 release kinetics assessment showed that BMP-2 AM showed a reduced initial burst and continuous release for 28 days. In vitro co-culture with canine Ad-MSCs showed an increase in mineralization and mRNA expression of osteogenic markers in the BMP-2 AM group compared with that of the BMP-2-adsorbed scaffold group. In vivo bone regeneration evaluation 12 weeks after surgery showed that the BMP-2 AM/PDA group exhibited the highest bone volume in the scaffold, followed by the BMP-2/PDA group. High cortical bone connectivity was observed in the PDA-coated scaffold groups. ConclusionThese findings suggest that the combined use of PDA-coated 3D-printed bone scaffolds and BMP-2 AM can successfully induce bone regeneration even in load-bearing bone segmental defects. The translational potential of this articleA 3D-printed PCL/β-TCP scaffold was fabricated to mimic the cortical bone of the femur. Along with the application of PDA surface modification and sustained BMP-2 release via AM, the developed scaffold could provide suitable osteoconduction, osteoinduction, and osteogenesis in both in vitro settings and in vivo rabbit femoral segmental bone defect models. Therefore, our findings suggest a promising therapeutic option for treating challenging long bone segmental defects, with potential for future clinical application.

A Chemometric Exploration of Potential Chemical Markers and an Assessment of Associated Risks in Relation to the Botanical Source of Fruit Spirits.

Chemometric evaluation of potentially harmful volatile compound and toxic metal(loid) distribution patterns in fruit spirits relating to distinct fruit classes most commonly used in spirit production highlighted the potential of several volatiles as candidates for differentiation markers while dismissing toxic metal(loid)s. Pome fruit and grape pomace spirits were mostly characterized by a lower abundance of n-propanol, methanol, ethyl acetate and acetaldehyde, while stone fruit spirits contained lower amounts of isoamyl alcohol and isobutanol. Chemometric analysis of the fruit spirit composition of aromatics identified additional potential markers characteristic for certain fruits-benzoic acid ethyl ester, benzyl alcohol, benzaldehyde, butanoic acid 3-methyl-ethyl ester, butanoic acid 2-methyl-ethyl ester and furfural. This study explored the variability in the risk potential of the investigated spirits, considering that some chemicals known to be detected in spirits are potent health hazards. Ethyl carbamate in combination with acetaldehyde showed a higher potential risk in stone fruit spirits, methanol in stone and pome fruit spirits and acetaldehyde in grape pomace spirits. It is of great interest to evaluate to what extent consumers' preference for spirits of distinct fruit types affects health risks. Consumers of stone fruit spirits are potentially at higher risk than those consuming pome fruit or grape pomace spirits.

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

Trehalose Prevents IL-4/IL-13–Induced Skin Barrier Impairment by Suppressing IL-33 Expression and Increasing NRF2 Activation in Human Keratinocytes In Vitro

Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis (AD). At high concentrations, the nonreducing disaccharide α-d-glucopyranosyl α-d-glucopyranoside (trehalose) induces a transient senescence-like state in fibroblasts and promotes wound repair. Here, we investigated the effect of trehalose on normal human keratinocytes (KCs) and demonstrated its specific role in the skin barrier. RNA-seq analysis revealed that trehalose regulates the expression of many skin-barrier-associated genes. T helper 2 (Th2) cytokines interleukin (IL)-4/IL-13 were observed to downregulate several differentiation markers (FLG, LOR, K1, and K10) and epidermal antimicrobial proteins in monolayer-cultured KCs and living skin equivalents (LSE), and impaired skin barrier function in LSE, all of which were significantly upregulated or restored by trehalose. Trehalose inhibited IL-33 expression and reduced nuclear IL-33 levels by activating MEK5-extracellular signal-regulated kinase 5 (ERK5) and suppressing MEK1/2-ERK pathway. It also increased nuclear factor erythroid 2-related factor 2 (Nrf2) activation to trigger antioxidant enzyme production via c-Jun N-terminal kinase (JNK), thus, neutralizing IL-4/IL-13-mediated oxidative stress. Trehalose prevented IL-4/IL-13-mediated signal transducer and activator of transcription (STAT)3/STAT6 activation and restored IL-4/IL-13-suppressed skin barrier molecules via IL-33 downregulation and Nrf2 activation. This study demonstrated that trehalose may play a role in skin barrier repair in AD.

Enzymatic activity of fibroblast activation protein-α is essential for TGF-β1-induced fibroblastic differentiation of human periodontal ligament cells

Human periodontal ligament cells (hPDLCs) contain multipotent postnatal stem cells that can differentiate into PDL fibroblasts, osteoblasts, and cementoblasts. Interaction between the extracellular environment and stem cells is an important factor for differentiation into other progenitor cells. To identify cell surface molecules that induce PDL fibroblastic differentiation, we developed a series of monoclonal antibodies against membrane/ECM molecules. One of these antibodies, an anti-PDL25 antibody, recognizes approximately a 100 kDa protein, and this antigenic molecule accumulates in the periodontal ligament region of tooth roots. By mass spectrometric analysis, we found that the antigenic molecule recognized by the anti-PDL25 antibody is fibroblast activation protein α (FAPα). The expression level of FAPα/PDL25 increased in TGF-β1-induced PDL fibroblasts, and this protein was localized in the cell boundaries and elongated processes of the fibroblastic cells. Ectopic expression of FAPα induced fibroblastic differentiation. In contrast, expression of representative markers for PDL differentiation was decreased by knock down and antibody blocking of FAPα/PDL25. Inhibition of dipeptidyl peptidase activity by a potent FAPα inhibitor dramatically inhibited PDL fibroblastic marker expression but did not affect in cell proliferation and migration.

Association of the Triglyceride-Glucose Index and Related Metabolites with sICAS of the Posterior Circulation

ObjectiveThe triglyceride-glucose index (TyG), a reliable marker of insulin resistance (IR), has an unclear association with symptomatic intracranial atherosclerotic stenosis in the posterior circulation (POC-sICAS). This study aimed to investigate the association between the TyG index, associated metabolites, and POC-sICAS. MethodsA total of 106 patients with POC-sICAS and 81 with asymptomatic intracranial atherosclerotic stenosis of the posterior circulation (POC-aICAS) were retrospectively enrolled. The TyG index was calculated using the formula: ln [fasting triglyceride (mg/dL) × fasting blood glucose (mg/dL)/2]. Logistic regression analysis evaluated the correlation between the TyG index and POC-sICAS. Differential metabolic markers were identified using liquid chromatography-tandem mass spectrometry. The predictive value of the TyG index and differential metabolites for POC-sICAS was assessed through receiver operating characteristic (ROC) curve analysis. ResultsPatients with POC-sICAS had a significantly higher TyG index than those with POC-aICAS (9.25 [interquartile range {IQR} 8.79-9.92] vs 8.87 [IQR 8.83-9.26]; P<0.001). The TyG index was an independent risk factor for POC-sICAS (adjusted odds ratio [OR]: 2.20, 95% confidence interval [CI]: 1.09-4.42, P=0.027), particularly in patients with diabetes mellitus (adjusted OR: 14.39, 95% CI: 2.37-87.36, P=0.004). The association was stronger in patients younger than 65 years with a high TyG index (≥8.99) (adjusted OR: 4.35, 95% CI: 1.12-16.94, P=0.034). The TyG index positively correlated with 90-day poor prognosis in those with POC-sICAS (R=0.4042, P=0003), especially in those with diabetes mellitus (R=6405, P<00001) and those younger than 65 years (R=0.4628, P=0002). Moreover, 11 differential metabolites were significantly related to the TyG index. The TyG index combined with pregnanediol showed significant discrimination between patients with POC-sICAS and POC-aICAS (area under the curve [AUC]=1, 95% CI 1.000-1.000; P<0.001). ConclusionsThe TyG index and pregnanediol could help identify patients with a high risk of ischemic stroke in POC-ICAS. Moreover, the TyG index was associated with a poor 90-day prognosis in patients with POC-sICAS.

CD56briCD38+ as a novel neutrophil-specific marker in chronic myeloid leukemia

BackgroundChronic myeloid leukemia (CML) is classified as a subtype of myeloproliferative neoplasm, and bone marrow flow cytometry does not reveal any specific immunophenotype. Interestingly, aberrant expression of cluster of differentiation (CD) markers such as CD56 and CD38 has been observed on neutrophils in CML patients. Therefore, we investigated the abnormal expression of CD56 and CD38 in CML neutrophils to explore their diagnostic value in identifying CML through flow cytometry. MethodsWe have developed a multi-parameter flow cytometry assay to identify aberrant immunophenotypes in CML neutrophils among bone marrow nucleated cells. ResultsCompared to healthy donors and patients with a reactive neutrophilia or other hematological malignancies, the percentage of CD56briCD38+ neutrophil subsets in CML patients exhibits a distinctive increase (cut-off value, 2.0%). The specificity and sensitivity associated with the learning cohort (168 samples) were 90.8% and 84.9%, respectively, while in the validation cohort (194 samples), they were 90.7% and 84.7%. The accumulation of CD56briCD38+ neutrophil subsets, which demonstrate are abnormal characteristics, is independent of the neutrophil count, BCR/ABL1 fusions and risk stratification but associated with blast cells immunophenotype. Moreover, this increase disappears in CML patients after treatment with tyrosine kinase inhibitors when the curative effect was satisfactory. ConclusionsWe conclude that an increase in the proportion of CD56briCD38+ neutrophil subsets exceeding 2.0% of total neutrophils serves as a highly sensitive and specific flow cytometry marker, enabling rapid and accurate identification of CML.

Facial pore refining by targeting dermal and epidermal functions: Assessment across age and gender.

Conspicuous facial pores are benign but represent a cosmetic concern for men and women. Recent works described dermal and epidermal impairments as clinical causes of enlarged pores. Morphological modifications of skin at the site of pores were associated with collagen density loss, possible alteration of extracellular matrix and abnormal differentiation of keratinocytes. A composition containing mannose-6-phosphate (Active Complex) was designed to address these different aspects of pore enlargement. Invitro and exvivo evaluations were conducted in different models mimicking disturbance of dermal and epidermal functions. The pore refining activity of Active Complex was assessed in two clinical trials studying a Caucasian women cohort and an Asian men cohort. At the dermal level, Active Complex upregulated collagen I and decorin synthesis, and genes encoding collagens I, III, V, VII, XVII; suggesting its ability to favor collagen fiber organization and anchorage. The downregulation of matrix metalloprotease, involved in extracellular matrix degradation, reinforced the protective effect of Active Complex in the dermis. Active Complex down modulated differentiation markers in keratinocytes as well as genes involved in cell renewal. Study of reconstructed human epidermis modeling keratinocyte hyperproliferation revealed that Active Complex mitigated two markers of this state: number of nuclei in the stratum corneum and involucrin expression. Clinical trials confirmed the pore refining activity of Active Complex on men and women of different ages and ethnicities; -24% total skin pore area after 56 days of application on women, and -30.2% on men after 7 days. This work demonstrates the interest to target dermal and epidermal modifications described in conspicuous pore area, especially dermis fiber organization, to address this cosmetic concern.

Evaluation of Potential Roles of Zinc Finger Homeobox 3 (Zfhx3) Expressed in Chondrocytes and Osteoblasts on Skeletal Growth in Mice.

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function forZinc Finger Homeobox 3(Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked outZfhx3conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice withZfhx3deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblastZfhx3conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass.

Vδ1 T Cells Integrated in Full-Thickness Skin Equivalents: A Model for the Role of Human Skin–Resident γδT Cells

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

Clustered ARPE-19 cells distinct in mitochondrial membrane potential may play a pivotal role in cell differentiation

Age-related macular degeneration (AMD) is associated with the dysfunction and degeneration of retinal pigment epithelium (RPE) cells. Here, we examined how the formation and expansions of cell clusters are regulated by the differentiation of the RPE cells. In this study, ARPE-19 cells were cultivated in standard or differentiation media, i.e., without or with nicotinamide, to evaluate the spreading of cell clusters specified with differentiated cell phenotypes. Mitochondria membrane potential (MMP) and the distribution of the RPE cell clusters was also monitored with or without rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor. Cultured ARPE-19 cells generated scattered cell clusters composed mostly of smaller size cells expressing the differentiation markers mouse anti-cellular retinaldehyde–binding protein (CRALBP) and Bestrophin only in differentiation medium. After the increase of the number of clusters, the clusters appeared to paracellularly merge, resulting in expansion of the area occupied by the clusters. Of note, the cells within the clusters selectively had high MMP and were in accordance with the expression of RPE differentiation markers. Rotenone repressed the formation of the clusters and decreased intracellular MMP. The above results suggest that clustering of RPE cells with functional mitochondria plays a pivotal role in RPE cell differentiation process and the ETC complex I inhibition greatly influences the composition of RPE cells that are degenerated or differentiation disposed.