Overexpressing S100A9 ameliorates NK cell dysfunction in estrogen receptor-positive breast cancer.

BACKGROUND: Estrogen receptor (ER) positive human epidermal growth factor receptor 2 (HER2) negative breast cancer (ER+/HER2-BC) and triple-negative breast cancer (TNBC) are two distinct breast cancer molecular subtypes, especially in tumor immune microenvironment (TIME). The TIME of TNBC is considered to be more inflammatory than that of ER+/HER2-BC. Natural killer (NK) cells are innate lymphocytes that play an important role of tumor eradication in TME. However, studies focusing on the different cell states of NK cells in breast cancer subtypes are still inadequate. METHODS: In this study, single-cell mRNA sequencing (scRNA-seq) and bulk mRNA sequencing data from ER+/HER2-BC and TNBC were analyzed. Key regulator of NK cell suppression in ER+/HER2-BC, S100A9, was quantified by qPCR and ELISA in MCF-7, T47D, MDA-MB-468 and MDA-MB-231 cell lines. The prognosis predictability of S100A9 and NK activation markers was evaluated by Kaplan-Meier analyses using TCGA-BRAC data. The phenotype changes of NK cells in ER+/HER2-BC after overexpressing S100A9 in cancer cells were evaluated by the production levels of IFN-gamma, perforin and granzyme B and cytotoxicity assay. RESULTS: By analyzing scRNA-seq data, we found that multiple genes involved in cellular stress response were upregulated in ER+/HER2-BC compared with TNBC. Moreover, TLR regulation pathway was significantly enriched using differentially expressed genes (DEGs) from comparing the transcriptome data of ER+/HER2-BC and TNBC cancer cells, and NK cell infiltration high/low groups. Among the DEGs, S100A9 was identified as a key regulator. Patients with higher expression levels of S100A9 and NK cell activation markers had better overall survival. Furthermore, we proved that overexpression of S100A9 in ER+/HER2-cells could improve cocultured NK cell function. CONCLUSION: In conclusion, the study we presented demonstrated that NK cells in ER+/HER2-BC were hypofunctional, and S100A9 was an important regulator of NK cell function in ER+BC. Our work contributes to elucidate the regulatory networks between cancer cells and NK cells and may provide theoretical basis for novel drug development.

Comparing survival outcomes between neoadjuvant and adjuvant chemotherapy within T2N1M0 stage hormone receptor-positive, HER2-negative breast cancer: a retrospective cohort study based on SEER database.

BACKGROUND: Guideline recommendations for the application of neoadjuvant chemotherapy (NACT) in T2N1M0 stage hormone receptor-positive, HER2-negative (HR + /HER2-) breast cancer are ambiguous. The debate continues regarding whether NACT or adjuvant chemotherapy (ACT) offers superior survival outcomes for these patients. MATERIALS AND METHODS: Female patients diagnosed with HR + /HER2- breast cancer at T2N1M0 stage between 2010 and 2020, were identified from the Surveillance, Epidemiology, and End Results database and divided into two groups, the NACT group and the ACT group. Propensity score matching (PSM) was utilized to establish balanced cohorts between groups, considering baseline features. Kaplan-Meier (K-M) analysis and the Cox proportional hazards model were executed to assess the efficacy of both NACT and ACT in terms of overall survival (OS) and breast cancer-specific survival (BCSS). A logistic regression model was employed to examine the association between predictive variables and response to NACT. RESULTS: After PSM, 4,682 patients were finally included. K-M curves showed that patients receiving NACT exhibited significantly worse OS and BCSS when compared with patients undergoing ACT. Multivariable Cox analysis indicated that not achieving pathologic complete response (non-pCR) after NACT (versus ACT), was identified as an adverse prognostic factor for OS (HR 1.58, 95% CI 1.36-1.83) and BCSS (HR 1.70, 95% CI 1.44-2. 02). The logistic regression model revealed that low tumor grade independently predicted non-pCR. CONCLUSION: Among T2N1M0 stage HR + /HER2- patients, OS and BCSS of NACT were inferior to ACT. Patients who attained non-pCR after NACT demonstrated significantly worse survival outcomes compared with those who received ACT.

Identification of a PANoptosis-related Gene Signature for Predicting the Prognosis, Tumor Microenvironment and Therapy Response in Breast Cancer.

Breast cancer (BC) is the most prevalent malignancy among women worldwide. Mounting evidence suggests that PANoptosis participates in cancer development and therapy. However, the role of PANoptosis in BC remains unclear. In this study, we identified ten PANoptosis-related genes using Cox regression analysis, random forest (RF) algorithm and least absolute shrinkage and selection operator (LASSO) algorithm. A PANoptosis-related score (PRS) was calculated based on the coefficient of LASSO. Notably, we divided the patients into high- and low-risk groups according to the PRS and revealed a negative correlation between PRS and overall survival. Next, a nomogram model was constructed and validated to improve the clinical application of PRS. Functional enrichment analyses and the Bayesian network demonstrated that differentially expressed genes between high- and low-risk groups were mainly enriched in immune-related pathways. Besides, we found significant differences in tumor mutation burden and tumor immune microenvironment between patients in these two groups using bulk-RNA and single-cell RNA sequencing data. Furthermore, charged multivesicular body protein 2B (CHMP2B) was identified as the hub gene by combining LASSO, weighted gene co-expression network analysis, RF and eXtreme Gradient Boosting. Importantly, using immunohistochemistry analysis based on our tissue microarray, we found that CHMP2B was highly expressed in tumor tissue, and CD4 and CD8 were more likely to be positive in the CHMP2B-negative group. Survival analyses revealed that CHMP2B adversely impacted the survival of BC patients. In conclusion, we not only constructed a highly accurate predictive model based on PRS, but also revealed the importance of PANoptosis-related gene signature in the modulation of the tumor microenvironment and drug sensitivity in BC.

BPIFB1 promotes metastasis of hormone receptor-positive breast cancer via inducing macrophage M2-like polarization.

Metastasis is an important factor affecting the prognosis of hormone receptor-positive breast cancer (BC). However, the molecular basis for migration and invasion of tumor cells remains poorly understood. Here, we identify that bactericidal/permeability-increasing-fold-containing family B member 1 (BPIFB1), which plays an important role in innate immunity, is significantly elevated in breast cancer and associated with lymph node metastasis. High expression of BPIFB1 and its coding mRNA are significantly associated with poor prognosis of hormone receptor-positive BC. Using enrichment analysis and constructing immune infiltration evaluation, we predict the potential ability of BPIFB1 to promote macrophage M2 polarization. Finally, we demonstrate that BPIFB1 promotes the metastasis of hormone receptor-positive BC by stimulating the M2-like polarization of macrophages via the establishment of BC tumor cells/THP1 co-culture system, qPCR, Transwell assay, and animal experiments. To our knowledge, this is the first report on the role of BPIFB1 as a tumor promoter by activating the macrophage M2 polarization in hormone receptor-positive breast carcinoma. Together, these results provide novel insights into the mechanism of BPIFB1 in BC.

Inhibition of lncRNA PCAT19 promotes breast cancer proliferation.

BACKGROUND: Breast cancer (BC) is the most common malignancy affecting women. It is vital to explore sensitive biological markers to diagnose and treat BC patients. Recent studies have proved that long noncoding RNAs (lncRNAs) were involved in breast tumor progression. Nonetheless, whether lncRNA prostate cancer-associated transcript 19 (PCAT19) impacts BC development remains unknown. METHODS: We performed various bioinformatic analyses, including machine learning models to identify critical regulatory lncRNAs affecting prognosis in BC. The in situ hybridization (ISH) assay was carried out to confirm the expression levels of lncRNA PCAT19 in tissue specimens. MTT assay, wound healing assay, and transwell assay were performed to investigate PCAT19's impact on proliferation, migration, and invasion of BC cells. Mouse xenografts were used to examine the proliferation-inhibiting function of PCAT19 in vivo. RESULTS: Among the prognosis-associated lncRNAs, PCAT19 predicted a favorable prognosis in BC. Patients with high expression levels of PCAT19 had a lower clinical stage and less lymph node metastasis. The PCAT19-related genes were enriched in signaling pathways involved in tumor development, indicating PCAT19 was an essential regulator of BC. Using the ISH assay, we confirmed the expression level of lncRNA PCAT19 in human BC tissues was lower than normal breast tissues. Moreover, the knockdown of PCAT19 further confirmed its inhibiting ability in BC cell proliferation. Correspondingly, overexpressing PCAT19 reduced tumor size in mouse xenografts. CONCLUSIONS: Our study demonstrated that lncRNA PCAT19 suppressed the development of BC. PCAT19 might be a promising prognostic biomarker, which provides new insights into risk stratification for BC patients.

Construction of an immunogenic cell death-based risk score prognosis model in breast cancer.

Immunogenic cell death (ICD) is a form of regulated cell death that elicits immune response. Common inducers of ICD include cancer chemotherapy and radiation therapy. A better understanding of ICD might contribute to modify the current regimens of anti-cancer therapy, especially immunotherapy. This study aimed to identify ICD-related prognostic gene signatures in breast cancer (BC). An ICD-based gene prognostic signature was developed using Lasso-cox regression and Kaplan-Meier survival analysis based on datasets acquired from the Cancer Genome Atlas and Gene Expression Omnibus. A nomogram model was developed to predict the prognosis of BC patients. Gene Set Enrichment Analysis (GESA) and Gene Set Variation Analysis (GSVA) were used to explore the differentially expressed signaling pathways in high and low-risk groups. CIBERSORT and ESTIMATE algorithms were performed to investigate the difference of immune status in tumor microenvironment of different risk groups. Six genes (CALR, CLEC9A, BAX, TLR4, CXCR3, and PIK3CA) were selected for construction and validation of the prognosis model of BC based on public data. GSEA and GSVA analysis found that immune-related gene sets were enriched in low-risk group. Moreover, immune cell infiltration analysis showed that the immune features of the high-risk group were characterized by higher infiltration of tumor-associated macrophages and a lower proportion of CD8+ T cells, suggesting an immune evasive tumor microenvironment. We constructed and validated an ICD-based gene signature for predicting prognosis of breast cancer patients. Our model provides a tool with good discrimination and calibration abilities to predict the prognosis of BC, especially triple-negative breast cancer (TNBC).

Survival Outcomes of Breast-Conserving Therapy versus Mastectomy in Early-Stage Breast Cancer, Including Centrally Located Breast Cancer: A SEER-Based Study.

Purpose: This study aims to analyze the survival outcomes of breast cancer (BC) patients, especially centrally located breast cancer (CLBC) patients undergoing breast-conserving therapy (BCT) or mastectomy. Methods: Surveillance, epidemiology, and end results (SEER) data of patients with T1-T2 invasive ductal or lobular breast cancer receiving BCT or mastectomy were reviewed. We used X-tile software to convert continuous variables to categorical variables. Chi-square tests were utilized to compare baseline information. The multivariate logistic regression model was performed to evaluate the relationship between predictive variables and treatment choice. Survival outcomes were visualized by Kaplan-Meier curves and cumulative incidence function curves and compared using multivariate analyses, including the Cox proportional hazards model and competing risks model. Propensity score matching was performed to alleviate the effects of baseline differences on survival outcomes. Result: A total of 180,495 patients were enrolled in this study. The breast preservation rates fluctuated around 60% from 2000 to 2015. Clinical features including invasive ductal carcinoma (IDC), lower histologic grade, smaller tumor size, fewer lymph node metastases, positive ER and PR status, and chemotherapy use were independently correlated with BCT in both BC and CLBC cohorts. In all the classic Cox models and competing risks models, BCT was an independent favorable prognostic factor for BC, including CLBC patients in most subgroups. In addition, despite the low breast-conserving rate compared with tumors located in the other areas, CLBC did not impair the prognosis of BCT patients. Conclusion: BCT is optional and preferable for most early-stage BC, including CLBC patients.

LncRNA MBNL1-AS1 Represses Proliferation and Cancer Stem-Like Properties of Breast Cancer through MBNL1-AS1/ZFP36/CENPA Axis.

Background: Emerging studies have revealed long noncoding RNAs (lncRNAs) were key regulators of cancer progression. In this research, the expression and roles of MBNL1-AS1 were explored in breast cancer (BC). Methods: In this study, the MBNL1-AS1 expression in breast cancer tissue, as well as in cell line, was studied by qRT-PCR assays. The effects of MBNL1-AS1 on proliferation and stemness were evaluated by MTT assays, colony formation assays, orthotopic breast tumor mice models, extreme limiting dilution analysis (ELDA), fluorescence in situ hybridization (FISH), flow cytometry assays, and sphere formation assays. Flexmap 3D assays were performed to show that MBNL1-AS1 downregulated the centromere protein A (CENPA) secretion in BC cells. Western blot, RNA pull-down assays, RNA immunoprecipitation (RIP) assays, and FISH were conducted to detect the mechanism. Results: The results showed that the expression levels of MBNL1-AS1 were downregulated in breast cancer tissues and cell lines. In vitro and in vivo studies demonstrated that overexpression of MBNL1-AS1 markedly inhibited BC cells proliferation and stemness. RNA pull-down assay, RIP assay, western blot assay, and qRT-PCR assay showed that MBNL1-AS1 downregulated CENPA mRNA via directly interacting with Zinc Finger Protein 36 (ZFP36) and subsequently decreased the stability of CENPA mRNA. Restoration assays also confirmed that MBNL1-AS1 suppressed the CENPA-mediated proliferation and stemness in breast cancer cells. Conclusions: The new mechanism of how MBNL1-AS1 regulates BC phenotype is elucidated, and the MBNL1-AS1/ZFP36/CENPA axis may be served as a therapeutic target for BC patients.

Sudden cardiac death of Duchenne muscular dystrophy with NT-proBNP in pericardial fluid as a useful biomarker for diagnosis of the cause of death: a case report.

Duchenne muscular dystrophy (DMD) is one of the most common and severest muscular dystrophies. Although it can be a cause of death when associated with cardiac muscle and/or respiratory muscles, no cases of sudden deaths in the setting of undiagnosed DMD with cardiac involvement have been reported in the literatures. Previous studies showed that N-terminal-proBNP (NT-proBNP) was a robust laboratory biomarker to diagnose and monitor cardiac failure in clinical situations, suggesting that it may be used as an auxiliary indicator for diagnosis on left ventricular dysfunction in sudden cardiac deaths in forensic settings. Here, we reported a case of 29-year-old man who died suddenly. Autopsy revealed that muscles of the body were almost replaced by fatty and fibrotic tissues. The heart was enlarged with disarray and degeneration of cardiomyocytes in cardiac muscle. Total absence of dystrophin was detected by immunohistochemical staining, which confirmed DMD. Postmortem biochemical test of pericardial fluid revealed a high level of NT-proBNP, indicating dysfunction of the left ventricle before death. The cause of death was certified as an early dilated cardiomyopathy (DCM)/dysfunction of the left ventricle secondary to DMD, suggesting that sudden cardiac death with cardiac dysfunction could be identified by immunohistochemical method in combination with pericardial fluid NT-proBNP determination after systemic autopsy.

Conformal Shell Amorphization of Nanoporous Ag-Bi for Efficient Formate Generation.

Simultaneous attainments of high conductivity and superior catalysis are major challenges for amorphous electrocatalysts in carbon dioxide electroreduction at high overpotential. In this study, one protocol is first demonstrated to drive the shell amorphization of nanoporous Ag-Bi (a-NPSB) catalyst with the spatially interconnected ligament during the initial stage of CO2ER. This newborn a-NPSB bestows the outstanding catalysis, evidenced by a Faradaic efficiency of 88.4% for formate production at -1.15 V vs RHE, specific current density of 21.2 mA cm-2, and mass specific current density of 321 mA mg-1. The unique catalysis is considered as the collective contribution of the conductive ligament internally and amorphous Bi2O3 shell with about 3.2 nm thickness externally. Simultaneous obtaining of the conductivity of inner metals and catalytic activity of the amorphous shell will pave a new avenue for designing a robust electrode during electrochemical reaction.

Activation of cannabinoid type 2 receptor protects skeletal muscle from ischemia-reperfusion injury partly via Nrf2 signaling.

AIMS: Cannabinoid type 2 (CB2) receptor activation has been shown to attenuate IRI in various organs. NF-E2-related factor (Nrf2) is an anti-oxidative factor that plays multiple roles in regulating cellular redox homeostasis and modulating cell proliferation and differentiation. The protective effects of CB2 receptor activation on skeletal muscle IRI and the underlying mechanism that involves Nrf2 signaling remain unknown. MAIN METHODS: We evaluated the in vivo effect of CB2 receptor activation by the CB2 receptor agonist AM1241 on IR-induced skeletal muscle damage and early myogenesis. We also assessed the effects of CB2 receptor activation on C2C12 myoblasts differentiation and H2O2-induced C2C12 myoblasts damage in vitro, with a focus on the mechanism of Nrf2 signaling. KEY FINDINGS: Our results showed that CB2 receptor activation reduced IR-induced histopathological lesions, edema, and oxidative stress 1 day post-injury and accelerated early myogenesis 4 days post-injury in mice. Nrf2 knockout mice that were treated with AM1241 exhibited deteriorative skeletal muscle oxidative damage and myogenesis. In vitro, pretreatment with AM1241 significantly increased the expression of Nrf2 and its nuclear translocation, attenuated the decrease in H2O2-induced C2C12 cell viability, and decreased reactive oxygen species generation and apoptosis. CB2 receptor activation also significantly enhanced C2C12 myoblasts differentiation, which was impaired by silencing Nrf2. SIGNIFICANCE: Overall, CB2 receptor activation protected skeletal muscle against IRI by ameliorating oxidative damage and promoting early skeletal muscle myogenesis, which was partly via Nrf2 signaling.

Evaluation of specific neural marker GAP-43 and TH combined with Masson-trichrome staining for forensic autopsy cases with old myocardial infarction.

It has been a puzzling forensic task to determine the cause of death as a result of old myocardial infarction (OMI) in the absence of recognizable acute myocardial infarction. Recent studies indicated that the heterogeneous cardiac nerve sprouting and sympathetic hyperinnervation at border zones of the infarcted site played important roles in sudden cardiac death (SCD). So, the present study explored the value of growth associated protein-43 (GAP-43) and tyrosine hydroxylase (TH) as objective and specific neural biomarkers combined with Masson-trichrome staining for forensic autopsy cases. Myocardium of left ventricle of 58 medicolegal autopsy cases, 12 OMI cases, 12 acute/OMI cases, and 34 control cases, were immunostained with anti-GAP-43 and anti-TH antibodies. Immunoreactivity of GAP-43 and TH identified nerve fibers and vascular wall in OMI cases and acute/OMI cases. Specifically, TH-positive nerve fibers were abundant at border zones of the infarcted site. There were a few GAP-43 and TH expressions in the control cases. With Masson-trichrome staining, collagen fibers were blue and cardiac muscle fibers were pink in marked contrast with the surrounding tissue, which improved the location of nerve fibers. Thus, these findings suggest that immunohistochemical detection of GAP-43 and TH combined with Masson-trichrome staining can provide the evidence for the medicolegal expertise of SCD due to OMI, and further demonstrate a close relationship between sympathetic hyperinnervation and SCD.

Nanoporous Au-Ag shell with fast kinetics: integrating chemical and plasmonic catalysis.

Nanoporous noble metals and alloys are widely utilized as efficient catalysts, because they have high surface-to-volume ratios for sufficient active sites and induce molecule polarization through plasmon excitation as well. Herein, we demonstrate one approach to fabricate nanoporous Au-Ag shell. Such material represents the dual functions serving as efficient catalysts and high-performance surface-enhanced Raman scattering substrate. In situ spectrum acquisition can track the conversion of p-nitrothiophenol to 4, 4'-dimercapto-azobenzene at ambient temperature. In particular, as a result of chemical catalysis of Ag elements and strong plasmon-molecule coupling, catalytic kinetics of nanoporous Au-Ag shell is 79.2-123.8 times faster than Au nanoparticles (NPs), and 2.2-3.3 times faster than Ag NPs. This investigation offers a route to design superior catalysts to integrate chemical and plasmonic catalysis.

Time-dependent Expression of MMP-2 and TIMP-2 after Rats Skeletal Muscle Contusion and Their Application to Determine Wound Age.

The ability to determine vitality and estimate the survival period after a wound is critical in routine forensic practice. The mRNA levels of MMP-2 and TIMP-2 were examined using quantitative real-time RT-PCR to determine the age of a wound. Furthermore, the colocalization of them with Macrophage Marker, respectively, was detected by double immunofluorescence, and a standardized rat model of skeletal muscle contusion was established. In the antemortem contused groups, a large number of macrophages showed positive staining for MMP-2 and TIMP-2, and the expression of MMP-2 and TIMP-2 mRNA increased sharply at 3 days postinjury, with relative quantities of 5.75 and 2.98. No samples in the other groups showed relative quantities of >5.75 and 2.98; therefore, relative quantities exceeding 5.75 and 2.98 were strongly indicated 3 days after contusion. In addition, there was a significant decrease in the relative quantity in the postmortem contused groups, indicating that they were useful for diagnosing vitality.

Time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and its application for wound age determination.

OBJECTIVE: To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination. METHODS: A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. RESULTS: Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. CONCLUSION: The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.

Beneficial effects of cannabinoid receptor type 2 (CB2R) in injured skeletal muscle post-contusion.

The aim of the current study was to investigate the effects of cannabinoid receptor type 2 (CB2R) on the repair process of injured skeletal muscle, which could potentially lay solid foundations as a novel target for curing muscular fibrosis in future. A standardized rat model of skeletal muscle contusion was established, where rats were treated with the CB2R agonist JWH-133 or antagonist AM-630. The in vivo results revealed that CB2R activation with JWH-133 significantly diminished the fibrotic areas, down-regulated the mRNA levels of collagen type I/ІІІ and augmented the number of multinucleated regenerating myofibers in the injured zones. The reasons leading to the aforementioned results were directly attributable to decreased mRNA levels of TGF-ß1, FN-EIIIA and α-SMA, reduced accumulation of myofibroblasts, and concomitantly increased mRNA levels of matrix metalloproteinase-1/2. However, we observed contrasting changes in rats treated with the CB2R antagonist AM-630. These results revealed multiple effects of CB2R in systematically inhibiting fibrotic formation and improving muscle regeneration, alongside its potential for clinical application in patients with skeletal muscle injuries and diseases.

[Correlation between percentages of PMN, MNC, FBC and wound age after skeletal muscle injury in rats].

OBJECTIVE: To study the percentages of polymorphonuclear leukocytes (PMN), mononuclear cells (MNC) and fibroblastic cells (FBC) in different post-traumatic intervals after skeletal muscle mechanical injury in rats. METHODS: The rat model of skeletal muscle mechanical injury was established. The rats were divided into injured groups (6 h, 12 h, 1 d, 3 d, 7 d, 10 d and 14 d after injury) and control group. The percentages of PMN, MNC and FBC in different post-traumatic intervals after skeletal muscle mechanical injury were assessed with HE staining and image analysis. RESULTS: At post-injury 6-12h, the percentages of PMN and MNC infiltration appeared in injured sites and that of PMN reached peak. At 1 d, the percentage of MNC infiltration appeared and reached peak, while that of PMN decreased. At 3-7 d, the percentage of FBC gradually increased, while that of PMN and MNC decreased. At 10-14d, the percentage of FBC reached peak. CONCLUSION: The percentages of PMN, MNC and FBC in injured zones showed time-dependent changes, which might be used as reference index for determination of age of skeletal muscle injury.

[Progress in myofibroblast and its application in forensic medicine].

The myofibroblasts have dual characteristics of smooth muscle cells and fibroblasts. In repairing tissular wound, myofibroblasts are involved in fibrogenesis and remodeling the extracellular matrix of the fibrotic cascades reaction. The review describes the morphological characteristics and biological behaviors of myofibroblasts and the application of skin wound age determination, which may provide reference for research in forensic medicine.

Systematical method for polyacrylamide and residual acrylamide detection in cosmetic surgery products and example application.

INTRODUCTION: In this paper, the authors presented a case of acrylamide poisoning in a middle-aged woman who had underwent unsuccessful cosmetic surgery six years earlier. The victim was told that the product that had been injected into her face was Restylane®, which mainly contained sodium hyaluronate and was the first and only Food and Drug Administration (FDA)-approved dermal filler for lip enhancement in the USA for more than 20 years. Widespread facial infections occurred several years post-injection; finally, the victim had to undergo removal surgery. Acrylamide poisoning was strongly suspected based on the victim's clinical manifestation. The product that had been injected into the victim's face was probably polyacrylamide hydrogel-based product, which had been prohibited by the State Food and Drug Administration (SFDA) in China in 2006. To confirm this suspicion, a systematical method was established to differentiate varieties of cosmetic surgery products and identify residential acrylamide. METHODS: The removed objects, original products and a certified reference sample of Restylane® were collected for examination. A direct microscopic examination was applied as a rapid screening method. Fourier transform infrared (FTIR) microspectroscopy analysis was subsequently performed to distinguish the main components from each sample. Automated solid phase extraction, ultra high performance liquid chromatography (SPE UHPLC) analysis was ultimately utilised and optimised to detect the residual acrylamide. Chromatographic separation was achieved on an ACQUITY UHPLC HSS T3 column. The mobile phase consisted of 0.01% aqueous formic acid solution and acetonitrile. The tunable UV (TUV) detection wavelength was at 202nm. RESULTS: The microscopic examination indicated that different samples had different morphological characteristics, depending on their main components. The FTIR spectrum showed that different polymers could be distinguished according to the CO stretching vibration (1655cm(-1)), NH bending vibration (1540cm(-1)) and CO stretching vibration (1078 and 1045cm(-1)). The UHPLC results demonstrated that the calibration curve was linear in the range of 0.5-20.0µg/mL, with a correlation coefficient of 0.999. The average recoveries of the method were 99-107% with an RSD of 1.6-6.3%. The detection limit was 0.1µg/mL (S/N=3). The analytical time was 6min per sample. Acrylamide was detected in the allegedly Restylane® injection. CONCLUSIONS: This systematical method provides a rapid, accurate and sensitive determination of polyacrylamide and residual acrylamide. The microscopic and FTIR spectroscopic examinations help to verify the existence of polyacrylamide quickly and easily. The optimised SPE UHPLC-TUV method offers a simpler and more sensitive approach to confirm the amount of acrylamide, comparing to the methods in the literature.

Nrf1 is time-dependently expressed and distributed in the distinct cell types after trauma to skeletal muscles in rats.

Our goal was to elucidate the dynamic expression and distribution of the nuclear factor erythroid-derived factor 2-related factor 1 (Nrf1) by immunohistochemistry, Western blotting, and real-time PCR during wound healing of contused skeletal muscle in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male healthy rats. Samples were taken at 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury, respectively (5 rats in each posttraumatic interval). 5 rats were employed as control. A weak immunoreactivity of Nrf1 was observed in the sarcoplasm and nuclei of normal myofibers in control rats. Prominent immunostaining for Nrf1 was seen in a large number of polymorphonuclear cells, round-shaped mononuclear cells and spindle-shaped fibroblastic cells, and regenerated multinucleated myotubes in the injured tissue. Subsequently, neutrophils, macrophages and myofibroblasts were identified as expressing Nrf1 by double immunofluorescent procedures. By real-time PCR analysis, Nrf1 expression was up-regulated and peaked at inflammatory phase. The expression tendency was also confirmed by Western blot. In conclusion, Nrf1 is time-dependently expressed in certain cell types, such as neutrophils, macrophages, myofibroblasts and regenerated multinucleated myotubes, suggesting that Nrf1 may modulate oxidative stress response and regeneration after trauma to skeletal muscles.