Inubritantrimers A-D: Trimerized Sesquiterpenoid [4 + 2] Adducts Featuring a Distinctive Spiro-Polycyclic Scaffold from Inula britannica.

Inubritantrimer A (1), a trace trimerized sesquiterpenoid [4 + 2] adduct featuring an unusual exo-exo type spiro-polycyclic scaffold, together with three new endo-exo [4 + 2] adducts, inubritantrimers B-D (2-4), were discovered from the flowers of Inula britannica. Their structures were elucidated using 1D/2D NMR, X-ray diffraction, and ECD approaches. 1 is characterized as a novel exo-exo trimer, synthesized biogenetically from three sesquiterpenoid monomers, featuring a unique linkage of C-11/C-1', C-13/C-3' and C-13'/C-3″, C-11'/C-1″ through a two-step exo [4 + 2] cycloaddition process. Compounds 1-4 exhibited modest cytotoxicity against breast cancer cells with IC50 values in the range of 5.84-12.01 µM.

Combined Cyclin-Dependent Kinase Inhibition Overcomes MAPK/Extracellular Signal-Regulated Kinase Kinase Inhibitor Resistance in Plexiform Neurofibroma of Neurofibromatosis Type I.

MAPK/extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitors (MEKis) have recently achieved surprising success in treating unresectable plexiform neurofibromas (PNFs). However, few studies have investigated the mechanisms of MEKi resistance in patients with PNF. We determined the efficacy of six different MEKis for treating PNFs, explored drug resistance mechanisms, and identified potential combination therapies to overcome resistance. By screening drug efficacy among six MEKis in human NF1-deficient PNF cell lines, TAK-733 was found to reduce PNF cell viability the most. We then cultured the TAK-733‒resistant cells and explored the potential targets for further treatment. Both high-throughput drug screening and RNA sequencing analyses of MEKi-resistant PNF cells identified cyclin-dependent kinase inhibitors as potential agents for PNFs. Dinaciclib, a cyclin-dependent kinase inhibitor, showed synergistic effects on MEKi-resistant cells. Coadministration of dinaciclib and TAK-733 significantly reduced cell viability and inhibited sphere formation and colony formation. Dinaciclib did not affect MEK signaling but decreased the expression of several prosurvival proteins, including survivin and cyclin-dependent kinase 1, to induce apoptosis and inhibit mitosis. TAK-733/dinaciclib combination therapy induced tumor reduction in PNF patient‒derived xenografts mouse models. Therefore, the combination of MEKi and cyclin-dependent kinase inhibitor may be promising for treating inoperable PNFs, especially when drug resistance exists. Our findings provide evidence for future clinical trials with MEKi-resistant patients with PNF.

Reconstruction of Large Cervicofacial Defects With Expanded Island Superficial Temporal Artery Flaps and an 810-nm Diode Laser Hair Removal Technique.

BACKGROUND: Extensive cervicofacial reconstruction is challenging for plastic surgeons. Because of the location of the adjacent scalp flap nourished by the superficial temporal artery (STA), it can be a candidate for cervicofacial reconstruction. OBJECTIVES: This article aims to report a combined treatment of an expanded island STA flap and an 810-nm diode laser hair removal technique for extensive cervicofacial defects. METHODS: Between January 2015 and December 2018, 10 patients with lower face and neck scar contraction were reconstructed with a bilateral or unilateral expanded STA island flap and an 810-nm diode laser for hair removal in this retrospective study. Hair removal via the 810-nm laser was started when the injected volume reached the volume of the expander, with a fluence of 35 to 40 J/cm2 and a 1 to 2 Hz repetition rate. Before second-stage surgery, the hair reduction rate was assessed. Twelve months after surgery, the degree of epilation efficacy according to the satisfaction scale and Global Aesthetic Improvement Scale was evaluated. RESULTS: This study included 2 single-pedicle flaps and 8 double-pedicle flaps. The average size of the implanted expanders was 600 mL. The average injected volume was 1405 mL. Before second surgery, there was a 67.4% hair reduction rate. Twelve months after surgery, the results of Global Aesthetic Improvement Scale were very good (3), good (6), average (1), and poor (0). CONCLUSIONS: The expanded island STA flap and 810-nm diode laser technique may be a novel treatment option for severe face and neck aesthetic reconstruction.

[Antidepressant effect of acidic polysaccharides from Poria and their regulation of neurotransmitters and NLRP3 pathway].

The rats were exposed to chronic unpredictable mild stress(CUMS) and kept in separate cages for inducing depressive disorder, which was judged by behavioral indicators. The number and morphology of neurons in hippocampal CA3 area and prefrontal cortex were observed by hematoxylin-eosin(HE) staining. The levels of brain-derived neurotrophic factor(BDNF), 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), norepinephrine(NE), glutamic acid(GLU), interleukin-1ß(IL-1ß), interleukin-18(IL-18), and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay(ELISA). Real-time polymerase chain reaction(RT-PCR) and Western blot were conducted to determine the mRNA and protein expression levels of related molecules in NLRP3 pathway. The results showed that compared with the model group, acidic polysaccharides from Poria at the low-, medium-, and high-doses(0.1, 0.3 and 0.5 g·kg~(-1)·d~(-1)) all improved the depression-like behavior of rats, increased the number of neurons and the levels of BDNF, 5-HT, 5-HIAA, DA, and NE in the hippocampus, and reduced GLU and serum IL-1ß, IL-18, and TNF-α levels. The mRNA expression levels of ASC, caspase-1, IL-1ß, and IL-18 and the protein expression levels of NLRP3, ASC, caspase-1, IL-1ß, and IL-18 in each medication group were down-regulated, whereas the protein expression levels of pro-caspase-1, pro-IL-1ß, and pro-IL-18 were up-regulated. All these have indicated that acidic polysaccharides from Poria exerted the antidepressant effect possibly by regulating neurotransmitters and NLRP3 inflammasome signaling pathway.

Clinical comparison of tenosynovial giant cell tumors, synovial chondromatosis, and synovial sarcoma: analysis and report of 53 cases.

BACKGROUND: Tenosynovial giant cell tumors (TGCTs), synovial chondromatosis (SC), and synovial sarcoma (SS) exhibit similarities in clinical features and histochemical characteristics, and differential diagnosis remains challenging in clinical practice. METHODS: Data were collected from the pathology database of Shanghai Ninth People's Hospital regarding patients who underwent surgery from 2010 to 2019 with histologically confirmed TGCTs, SC, and SS. Demographic and clinicopathological data of these patients were reviewed. Immunohistochemistry staining of 14 different markers was performed. Correlation analyses of the prognoses were evaluated. RESULTS: A total of 26 patients with TGCTs (8 diffuse TGCTs and 18 localized TGCTs), 16 with SC, and 11 with SS were identified. Pain was the main symptom of patients with both TGCTs and SC, while a palpable mass was the most common symptom for patients with SS. In addition to clinical features, we identified vital risk factors for disease recurrence. The mean follow-up periods were 51, 39, and 14 months for TGCTs, SC, and SS, respectively. Younger patients with diffuse TGCTs or patients with a higher neutrophil/lymphocyte ratio (NLR) displayed a significantly higher frequency of recurrence. We also plotted receiver operating characteristic (ROC) curve analysis for age and NLR. The area under the ROC curve (AUC) was calculated and demonstrated the ability to distinguish recurrent from nonrecurrent cases. In addition, higher CD163 expression was linked to recurrent diffuse TGCT cases. CONCLUSIONS: These data indicated possible characteristics of different aspects of TGCTs, SC, and SS. Further clarification and understanding of these factors will help with differential clinical diagnosis and recurrent risk assessment.

Selection of internal references for RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines.

Real-time quantitative PCR (RT-qPCR) has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference gene selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the optimal reference genes for the relative quantitative analysis of RT-qPCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most frequently used internal reference gene, was significantly unstable between various cell lines. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. These recommended internal reference gene selections may improve the accuracy and reproducibility of RT-qPCR in gene expression analyses of NF1 related tumors.

A narrative review of the role of fibroblasts in the growth and development of neurogenic tumors.

Neurogenic tumors, a group of tumors arising from neurogenic elements, could theoretically appear in every region of human bodies wherever nerves exist. Patients with these tumors suffer from both physical and psychological problems. However, as a relatively rare tumor type, therapies are relatively scarce for these tumors due to the limited understanding of the underlying mechanisms. Recently, a tailored tumor microenvironment containing multiple types of nonneoplastic cells has been considered to play an essential role in tumor survival, growth, and metastasis. Fibroblasts are a crucial constituent of the tumor microenvironment and have been found to promote tumor growth via multiple mechanisms. However, the understanding of the pivotal role of fibroblasts in the tumorigenesis and development of the neurogenic tumors is still incomplete, and studies in this area show differences in rates of progression among different neurogenic tumor subtypes. Nevertheless, all these neural crest-originated neoplasms show some similarities in the tumor microenvironment, indicating that studies of one subtype of neurogenic tumor might assist in clarifying the underlying mechanisms of other subtypes. This review aims to provide current studies showing the impacts of fibroblasts on major benign/malignant subtypes of neurogenic tumors, including neurofibromatosis type 1, neuroblastomas, pheochromocytomas, and malignant peripheral nerve sheath tumors. Multiple related mechanisms such as the fibroblasts regulating the tumor inflammation, angiogenesis, metabolism, and microenvironment establishment have been studied up to present. Consistently, we focus on how studies on various subtypes of these neurogenic tumors contribute to the establishment of potential future directions for further studies in this area. Clarifying the underlying mechanisms by which fibroblasts promote the growth and metastasis of neurogenic tumors will indicate new therapeutic targets for further clinical treatment.

Protein Tyrosine Phosphatase Receptor S Acts as a Metastatic Suppressor in Malignant Peripheral Nerve Sheath Tumor via Profilin 1-Induced Epithelial-Mesenchymal Transition.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with over half of cases developed in the context of neurofibromatosis type 1. Surgical resection is the only effective therapy for MPNST. The prognosis is very dismal once recurrence or metastasis occurs. Epithelial-mesenchymal transition (EMT) is a key process of recurrence and metastasis involving reorganizations of the actin cytoskeleton and actin-binding proteins (ABP) play a non-negligible role. Protein tyrosine phosphatase receptor S (PTPRS), a tumor suppressor previously reported in colorectal cancer, hepatocellular carcinoma and head and neck cancer, is thought to mediate cell migration and invasion by downregulation of EMT. However, its role in MPNST remains unknown. In the present study, by using tissue microarray we demonstrated low expression of PTPRS was related to poor prognosis in MPNST. Knockdown of PTPRS in MPNST cell lines increased migration/invasion and EMT processes were induced with increased N-cadherin and decreased E-cadherin, which indicated PTPRS may serve as a tumor suppressor in MPNST. In addition, we tested all EMT related ABP and found profilin 1 was significantly elevated in PTPRS downregulated MPNST cell lines. As a member of actin-binding proteins, profilins are regulators of actin polymerization and contribute to cell motility and invasion, which have been reported to be responsible for EMT. Moreover, results showed that downregulation of profilin 1 could restore the EMT processes caused by PTPRS downregulation in vitro and in vivo. Furthermore, high expression of profilin 1 was significantly associated with dismal prognosis. These results highlighted PTPRS served as a potential tumor suppressor in the recurrence and metastasis of MPNST via profilin 1 induced EMT processes and it might provide potential targets for future clinical therapeutics.

NF1, Neurofibromin and Gene Therapy: Prospects of Next-Generation Therapy.

Neurofibromatosis type 1 [NF1] is an autosomal dominant genetic disorder affecting multiple organs. NF1 is well known for its various clinical manifestations, including café-au-late macules, Lisch nodules, bone deformity and neurofibromas. However, there is no effective therapy for NF1. Current therapies are aimed at alleviating NF1 clinical symptoms but not curing the disease. By altering pathogenic genes, gene therapy regulates cell activities at the nucleotide level. In this review, we described the structure and functions of neurofibromin domains, including GAP-related domain [GRD], cysteine-serine rich domain [CSRD], leucine-rich domain [LRD] and C-terminal domain [CTD], which respectively alter downstream pathways. By transfecting isolated sequences of these domains, researchers can partially restore normal cell functions in neurofibroma cell lines. Furthermore, recombinant transgene sequences may be designed to encode truncated proteins, which is functional and easy to be packaged into viral vectors. In addition, the treatment effect of gene therapy is also determined by various factors such as the vectors selection, transgene packaging strategies and drug administration. We summarized multiple NF1 gene therapy strategies and discussed their feasibility from multiple angles. Different protein domains alter the function and downstream pathways of neurofibromin.

Computed Tomography-Based Differentiation of Benign and Malignant Craniofacial Lesions in Neurofibromatosis Type I Patients: A Machine Learning Approach.

Background: Because neurofibromatosis type I (NF1) is a cancer predisposition disease, it is important to distinguish between benign and malignant lesions, especially in the craniofacial area. Purpose: The purpose of this study is to improve effectiveness in the diagnostic performance in discriminating malignant from benign craniofacial lesions based on computed tomography (CT) using a Keras-based machine-learning model. Methods: The Keras-based machine learning technique, a neural network package in the Python language, was used to train the diagnostic model on CT datasets. Fifty NF1 patients with benign craniofacial neurofibromas and six NF1 patients with malignant peripheral nerve sheath tumors (MPNSTs) were selected as the training set. Three validation cohorts were used: validation cohort 1 (random selection of 90% of the patients in the training cohort), validation cohort 2 (an independent cohort of 9 NF1 patients with benign craniofacial neurofibromas and 11 NF1 patients with MPNST), and validation cohort 3 (eight NF1 patients with MPNST, not restricted to the craniofacial area). Sensitivity and specificity were tested using validation cohorts 1 and 2, and generalizability was evaluated using validation cohort 3. Results: A total of 59 NF1 patients with benign neurofibroma and 23 NF1 patients with MPNST were included. A Keras-based machine-learning model was successfully established using the training cohort. The accuracy was 96.99 and 100% in validation cohorts 1 and 2, respectively, discriminating NF1-related benign and malignant craniofacial lesions. However, the accuracy of this model was significantly reduced to 51.72% in the identification of MPNSTs in different body regions. Conclusion: The Keras-based machine learning technique showed the potential of robust diagnostic performance in the differentiation of craniofacial MPNSTs and benign neurofibromas in NF1 patients using CT images. However, the model has limited generalizability when applied to other body areas. With more clinical data accumulating in the model, this system may support clinical doctors in the primary screening of true MPNSTs from benign lesions in NF1 patients.

Investigating the potential of LSKL peptide as a novel hypertrophic scar treatment.

Hypertrophic scar (HTS) is a common pathologic dermal fibroproliferative disease after skin injury. Transforming growth factor ß (TGF-ß) plays a central role in HTS formation and development. Thrombospondin-1 (TSP-1) activates latent TGF-ß by binding to latency-associated peptide-ß on TGF-ß structure. So far, LSKL peptide was shown to selectively antagonize TSP-1. In this study, TSP-1 was first confirmed to be highly expressed in HTS. LSKL peptide was proven to inhibit the overexpression of extracellular matrix and contractile ability of HTS fibroblasts. In vivo, LSKL could attenuate the thickness of HTS, distortion of collagen alignment and fibrogenesis. Results also demonstrated that LSKL peptide not only remarkably attenuated cell proliferation and migration, but also induced cell apoptosis of HTS fibroblasts. Western blot analysis further revealed that LSKL peptide significantly suppressed the phosphorylation of PI3K, AKT, and mTOR, while not affecting the phosphorylation of Smad2/3 and MEK/ERK. These findings suggested that LSKL might be a promising anti-fibrosis agent to HTS through PI3K/AKT/mTOR signaling pathway.

Evaluation and Factors of Quality of Life Among Patients With Neurofibromatosis Type 1-Associated Craniofacial Plexiform Neurofibromas.

OBJECTIVES: The aim was to evaluate overall quality of life (QOL) and investigate impact factors in Chinese neurofibromatosis type 1 (NF1) patients, particularly in those with craniofacial plexiform neurofibromas (pNFs). METHODS: The Impact of NF1 on quality of life (INF1-QOL) Questionnaire were completed from a department of plastic and reconstructive surgery by 27 patients. Patients were 3 to 49 years of age. The correlation between subdomains were calculated using Pearson correlation. The difference between groups were evaluated using Fisher exact t-test. P value <0.05 were considered significant. RESULTS: In age group of craniofacial pNFs, significant difference presented in cosmetic appearance, role and outlook on life and general QOL. Higher impact on general QOL in adults (6/8) than children (1/7) pointed to more impaired QOL in adults, as well as 2 subdomains including appearance, role and outlook on life. The patients who have more than 50 cutaneous neurofibromas (cNFs) (6/7) presented a significantly greater negative impact on the role and outlook of life. No statistically significant difference of QOL were detected between craniofacial and non-craniofacial pNFs patients. CONCLUSIONS: Age and cNFs were 2 main factors that have a negative impact on QOL in craniofacial pNFs patients. Adults reported lower QOL in cosmetic appearance, the role and outlook of life and general QOL. Patients with more than 50 cNFs reported more negative impact on the role and outlook of life. A multidiscipline management for these patients is required, including psychosocial intervention.

The impact of host immune cells on the development of neurofibromatosis type 1: The abnormal immune system provides an immune microenvironment for tumorigenesis.

The immune system plays an essential role in the development of tumors, which has been demonstrated in multiple types of cancers. Consistent with this, immunotherapies with targets that disrupt these mechanisms and turn the immune system against developing cancers have been proven effective. In neurofibromatosis type 1 (NF1), an autosomal dominant genetic disorder, the understanding of the complex interactions of the immune system is incomplete despite the discovery of the pivotal role of immune cells in the tumor microenvironment. Individuals with NF1 show a loss of the NF1 gene in nonneoplastic cells, including immune cells, and the aberrant immune system exhibits intriguing interactions with NF1. This review aims to provide an update on recent studies showing the bilateral influences of NF1 mutations on immune cells and how the abnormal immune system promotes the development of NF1 and NF1-related tumors. We then discuss the immune receptors major histocompatibility complex class I and II and the PD-L1 mechanism that shield NF1 from immunosurveillance and enable the immune escape of tumor tissues. Clarification of the latest understanding of the mechanisms underlying the effects of the abnormal immune system on promoting the development of NF1 will indicate potential future directions for further studies and new immunotherapies.