Chemical Investigation on the Volatile Part of the CO2 Supercritical Fluid Extract of Infected Aquilaria sinensis (Chinese Agarwood).

This work is focused on the characterization of the composition of a CO2 supercritical fluid extract of Aquilaria sinensis (Chinese agarwood) collected in the Dongguan area (China) and infected by mechanical methods. The constituents of this extract were analyzed by gas chromatography-mass spectrometry (GC-MS) and quantified accurately by gas chromatography with a flame ionization detector (GC-FID), using an internal reference and predicted response factors. Since a significant number of components of this extract remained non-identified after the initial GC-MS analysis of the whole extract, its fractionation by chromatography on silica gel helped to characterize several additional constituents by isolation and structural analysis by NMR spectroscopy. The main components are the classical agarwood chromones (Flindersia chromone and its mono-, di-, and trimethoxylated analogues (respectively, 11.01% and 0.11-4.02%) along with sesquiterpenic constituents typically found in agarwood essential oils, like baimuxinal (1.90%) and kusunol (1.24%), as well as less common selinane dialdehydes (1.58-2.27%) recently described in the literature. Moreover, the structure and stereochemistry of a new sesquiterpenic alcohol, 14ß,15ß-dimethyl-7αH-eremophila-9,11-dien-8ß-ol (0.67%), was determined unambiguously by the combination of structural analysis (NMR, MS), hemisynthesis, and total synthesis, leading to dihydrokaranone and a neopetasane epimer.

A randomized, controlled clinical study of low-molecular-weight heparin improving pregnancy outcomes in PCOS women undergoing IVF: study protocol.

BACKGROUND: Polycystic ovary syndrome (PCOS), an incidence of 10-15% in women of reproductive age, shows sex hormone disorders, luteal insufficiency, and the tendency of placental villus space thrombus. The incidence of early pregnancy loss in women with PCOS is three to eight times higher than that in non-PCOS women. PCOS women were reported in a pre-thrombotic state, which was manifested by accelerated thrombin production, increased PAI-1 activity, and fibrinogen. Other research also found an over-activated state of women with PCOS in immune system. Therefore, changing the prethrombotic state of PCOS through anticoagulation may be a new way to improve the adverse pregnancy outcome of PCOS. Low-molecular-weight heparin (LMWH) is the most common used anticoagulant drug in pregnancy, and it also was proposed for the prevention of recurrent abortion, although the application of LMWH in PCOS population during early pregnancy has not been reported. The objective of this study is to investigate the effect of LMWH on pregnancy outcomes after invitro fertilization-frozen embryo transfer (IVF-FET) in patients with polycystic ovary syndrome. METHODS: A total of 356 PCOS women aged between 20 and 38 years which prepared for IVF followed with FET will be enrolled in the study. The patients, from four different hospitals stratified by age and body mass index (BMI), will be randomly divided into the study group who will be treated with LMWH started on the day of progesterone transformation (hormone therapy) during FET cycle and the control group without additional medicine. Serum or urine hCG test will be given 14 days after embryo transfer to confirm biochemical pregnancy. If pregnancy is positive, LMWH+ hormone therapy/hormone therapy will be continued for another 2 weeks. Transvaginal ultrasonography will be performed 14 days later to confirm intrauterine pregnancy. The primary outcome is the ongoing pregnancy, which is defined as intrauterine live fetus with ultrasound after 12 weeks of gestation. DISCUSSION: This is the first study protocol to investigate the efficacy of LMWH as an adjuvant drug for IVF-FET outcomes in PCOS women, by comparing differences in ongoing pregnancy rate, clinical pregnancy rate, live birth rate, and early pregnancy loss rate between LMWH group and the control group. TRIAL REGISTRATION: ClinicalTrials.gov ChiCTR2000036527. Registered on August 24, 2020.

Safety analysis of immediate breast reconstruction with a deep inferior epigastric perforator (DIEP) flap in the post-COVID-19 era: a comparison between pre- and post-pandemic cohorts.

Background: The demand for immediate breast reconstruction with a deep inferior epigastric perforator (DIEP) flap is recovering as coronavirus disease 2019 (COVID-19) transitions from a pandemic to an endemic. This study sought to evaluate the safety of resuming DIEP flap reconstruction in the post-COVID-19 era. Methods: Consecutive breast cancer patients who underwent immediate breast reconstruction with a DIEP flap at the Comprehensive Breast Health Center, Ruijin Hospital were retrospectively included in the study. The patients were divided into a post-pandemic group (Group A) and a pre-pandemic group (Group B). The clinicopathological factors, surgical procedures, and rates of post-operative complications were compared between the two groups using the Mann-Whitney U test and Chi-squared test. Results: A total of 167 patients were included in the study, of whom 119 (71.3%) were in Group A and 48 (28.7%) were in Group B. The two groups had similar clinicopathological features, including age (P=0.988), body mass index (P=0.504), and tumor, node, metastasis (TNM) stage (P=0.932). The Group A patients were more likely to receive single perforator DIEP flap transplantation than the Group B patients (n=28, 22.8% vs. n=3, 5.8%, P=0.007). There was a numerical decrease in the mean operating time of Group A patients compared to Group B patients (9.82 vs. 10.12 hours, P=0.172). The mean length of stay after the surgery was significantly shorter after the pandemic than before the pandemic (11.2 vs. 14.3 days, P<0.001). The complication rates between the two groups were similar. Conclusions: This study provides evidence that resuming DIEP reconstruction is safe in the post-COVID-19 era.

Tumor size is associated with adjuvant chemotherapy benefit in T1N0M0 triple-negative breast cancer: a multicenter and propensity score matched analysis.

Background: Triple-negative breast cancer (TNBC) is characterized by aggressive phonotypes and relatively poor outcomes. There are controversies on the effect of adjuvant chemotherapy in small (T1N0M0) TNBCs, especially among T1a-b patients. This study evaluated the survival benefit of adjuvant chemotherapy and influential factors in T1N0M0 TNBC patients. Methods: All T1N0M0 TNBC patients were identified from the Shanghai Jiao Tong University Breast Cancer Database (SJTU-BCDB) between January 2009 and December 2021. Propensity score matched (PSM) was applied to create a matched cohort. We used Kaplan-Meier analysis and Cox regression models to evaluate the associations of adjuvant chemotherapy with breast cancer-free interval (BCFI) and overall survival (OS). Stratified analysis according to different influential factors was also performed. Results: In total, 1,113 T1N0M0 TNBC patients (297 T1a, T1b and 816 T1c) were enrolled, including 928 patients with adjuvant chemotherapy and 185 patients without adjuvant chemotherapy. After matching 441 patients by using PSM analysis, 294 patients with chemotherapy and 147 patients without chemotherapy were identified. Patients with or without chemotherapy had similar BCFI (P=0.241) and OS (P=0.509). However, regarding patients with different tumor sizes, adjuvant chemotherapy could significantly improve BCFI in T1c patients (5-year BCFI: 92.1% vs. 79.5%, P=0.035) but not in T1a-b patients (5-year BCFI: 93.6% vs. 94.6%, P=0.546). No significant difference in OS was observed among patients with different tumor sizes. Subgroup analysis found that only tumor size was significantly associated with adjuvant chemotherapy benefit in terms of BCFI (Pinteraction=0.021) and OS (Pinteraction=0.040). Conclusions: The survival benefit of adjuvant chemotherapy was significantly associated with tumor size in T1N0M0 TNBC. Benefit of adjuvant chemotherapy was found in T1c, but not in T1a-b patients. Our findings do not support the routine use of chemotherapy in patients with T1a-bN0 TNBC.

Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy.

Cancer-associated adipocytes (CAAs), one of the primary stromal components, exhibit intimate crosstalk and release multiple cell factors mediating local and systemic biological effects. However, the role of CAAs in the regulation of systemic immune responses and their potential value in the clinical treatment of triple-negative breast cancer (TNBC) are not well described. Transcriptome sequencing was performed on CAA and normal adipocyte (NA) tissues isolated from surgically resected samples from TNBC patients and healthy controls. Cytokines, including C-X-C motif chemokine ligand 8 (CXCL8, also known as IL-8), secreted from NAs and CAAs were compared by transcriptome sequencing and enzyme-linked immunosorbent assay (ELISA). Proliferation, migration and invasion assays were employed to analyze the role of CAAs and CAA-derived CXCL8 (macrophage inflammatory protein-2 (MIP2) as a functional surrogate in mice). TNBC syngraft models were established to evaluate the curative effect of targeting CXCL8 in combination with anti-PD-1 therapies. Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting (WB), polymerase chain reaction (PCR) array, flow cytometry, immunohistochemistry (IHC), and immunofluorescence (IF) were applied to analyze immune cell infiltration and epithelial-mesenchymal transition (EMT) markers. Specifically, we demonstrated that CAAs and CAA-derived CXCL8 played important roles in tumor growth, EMT, metastasis and tumor immunity suppression. CAA-derived CXCL8 remodeled the tumor immune microenvironment not only by suppressing CD4+ T and CD8+ T immune cell infiltration but also by upregulating CD274 expression in TNBC. The combination of targeting the CXCL8 pathway and blocking the PD-1 pathway synergistically increased the tumor immune response and inhibited tumor progression. Thus, our results highlight the molecular mechanisms and translational significance of CAAs in tumor progression and immune ecosystem regulatory effects and provide a better understanding of the potential clinical benefit of targeting CAA-derived CXCL8 in antitumor immunity and as a new therapeutic moiety in TNBC.

Higher consumption of animal organ meat is associated with a lower prevalence of nonalcoholic steatohepatitis.

Background: Animal organ meat (offal) is a food with high nutrient density that is popular in different parts of the world, but its relationship with nonalcoholic steatohepatitis (NASH) is unclear. We aimed to examine whether daily animal organ meat consumption is associated with the presence of NASH in individuals with nonalcoholic fatty liver disease (NAFLD). Methods: A total of 136 Chinese adults with biopsy-proven NAFLD were included. Definite NASH was defined as NAFLD activity score ≥4 and at least one point for steatosis, ballooning, and lobular inflammation. Daily animal organ meat consumption was estimated using a self-administered validated food frequency questionnaire. Logistic regression analysis was performed to assess the association between animal organ meat intake and liver disease severity. Results: The 136 participants (80.9% men) of the study had a mean ± standard deviation (SD) age of 39.0±12.5 years and body mass index of 27.4±3.6 kg/m2. Prevalence of definite NASH was 65.4%. Daily median organ meat consumption was 1.30 g/1,000 kcal. Animal organ meat consumption was inversely associated with the presence of NASH even after adjustment of demographics, lifestyle variables, metabolic and dietary factors, as well as liver fibrosis stage; adjusted-odds ratios (95% confidence intervals) for NASH were 0.15 (0.03, 0.69) for the highest tertile and 0.18 (0.05, 0.70) for the medium tertile, compared to the lowest (reference) tertile of animal organ meat intake (P value for trend =0.024). Conclusions: Our results suggest for the first time that higher animal organ meat consumption is associated with a lower prevalence of NASH in Chinese individuals with biopsy-proven NAFLD.

Targeting glutamine metabolic reprogramming of SLC7A5 enhances the efficacy of anti-PD-1 in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease that is characterized by metabolic disruption. Metabolic reprogramming and tumor cell immune escape play indispensable roles in the tumorigenesis that leads to TNBC. Methods: In this study, we constructed and validated two prognostic glutamine metabolic gene models, Clusters A and B, to better discriminate between groups of TNBC patients based on risk. Compared with the risk Cluster A patients, the Cluster B patients tended to exhibit better survival outcomes and higher immune cell infiltration. In addition, we established a scoring system, the glutamine metabolism score (GMS), to assess the pattern of glutamine metabolic modification. Results: We found that solute carrier family 7 member 5 (SLC7A5), an amino acid transporter, was the most important gene and plays a vital role in glutamine metabolism reprogramming in TNBC cells. Knocking down SLC7A5 significantly inhibited human and mouse TNBC cell proliferation, migration, and invasion. In addition, downregulation of SLC7A5 increased CD8+ T-cell infiltration. The combination of a SLC7A5 blockade mediated via JPH203 treatment and an anti-programmed cell death 1 (PD-1) antibody synergistically increased the immune cell infiltration rate and inhibited tumor progression. Conclusions: Hence, our results highlight the molecular mechanisms underlying SLC7A5 effects and lead to a better understanding of the potential benefit of targeting glutamine metabolism in combination with immunotherapy as a new therapy for TNBC.

N-terminal α-amino SUMOylation of cofilin-1 is critical for its regulation of actin depolymerization.

Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational modification (PTM) plays pivotal roles in modulating protein function. Cofilins are key regulators of actin cytoskeleton dynamics and are well-known to undergo several different PTMs. Here, we show that cofilin-1 is conjugated by SUMO1 both in vitro and in vivo. Using mass spectrometry and biochemical and genetic approaches, we identify the N-terminal α-amino group as the SUMO-conjugation site of cofilin-1. Common to conventional SUMOylation is that the N-α-SUMOylation of cofilin-1 is also mediated by SUMO activating (E1), conjugating (E2), and ligating (E3) enzymes and reversed by the SUMO deconjugating enzyme, SENP1. Specific to the N-α-SUMOylation is the physical association of the E1 enzyme to the substrate, cofilin-1. Using F-actin co-sedimentation and actin depolymerization assays in vitro and fluorescence staining of actin filaments in cells, we show that the N-α-SUMOylation promotes cofilin-1 binding to F-actin and cofilin-induced actin depolymerization. This covalent conjugation by SUMO at the N-α amino group of cofilin-1, rather than at an internal lysine(s), serves as an essential PTM to tune cofilin-1 function during regulation of actin dynamics.

LEARN algorithm: a novel option for predicting non-alcoholic steatohepatitis.

Background: There is an unmet need for accurate non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH). Since impedance-based measurements of body composition are simple, repeatable and have a strong association with non-alcoholic fatty liver disease (NAFLD) severity, we aimed to develop a novel and fully automatic machine learning algorithm, consisting of a deep neural network based on impedance-based measurements of body composition to identify NASH [the bioeLectrical impEdance Analysis foR Nash (LEARN) algorithm]. Methods: A total of 1,259 consecutive subjects with suspected NAFLD were screened from six medical centers across China, of which 766 patients with biopsy-proven NAFLD were included in final analysis. These patients were randomly subdivided into the training and validation groups, in a ratio of 4:1. The LEARN algorithm was developed in the training group to identify NASH, and subsequently, tested in the validation group. Results: The LEARN algorithm utilizing impedance-based measurements of body composition along with age, sex, pre-existing hypertension and diabetes, was able to predict the likelihood of having NASH. This algorithm showed good discriminatory ability for identifying NASH in both the training and validation groups [area under the receiver operating characteristics (AUROC): 0.81, 95% CI: 0.77-0.84 and AUROC: 0.80, 95% CI: 0.73-0.87, respectively]. This algorithm also performed better than serum cytokeratin-18 neoepitope M30 (CK-18 M30) level or other non-invasive NASH scores (including HAIR, ION, NICE) for identifying NASH (P value <0.001). Additionally, the LEARN algorithm performed well in identifying NASH in different patient subgroups, as well as in subjects with partial missing body composition data. Conclusions: The LEARN algorithm, utilizing simple easily obtained measures, provides a fully automated, simple, non-invasive method for identifying NASH.

Early pregnancy exposure of maternal triglyceride levels and its effects on birth weight.

OBJECTIVES: The aim of this study was to evaluate the early pregnancy exposure of maternal triglyceride (mTG) and its effects on birth weight, which was an important indicator for nutritional status of newborns, and even its long-term health. METHODS: A retrospective cohort study was designed to investigate the relationship between mTG in early pregnancy and birth weight. Totally 32,982 women who had a singleton pregnancy and underwent serum lipids screening during early pregnancy were included in this study. Logistic regressions were used to evaluate the correlations between mTG levels and small for gestational age (SGA) or large for gestational age (LGA), and the restricted cubic spline models were applied to explore the dose-response relationship. RESULTS: The increased mTG levels during early pregnancy decreased the risk of SGA and increased the risk of LGA. The high mTG (>90th, 2.05 mM) was showed associated with higher risk of LGA (AOR, 1.35; 95 %CI, 1.20 to 1.50), and lower risk of SGA (AOR, 0.78; 0.68 to 0.89). Lower risk of LGA (AOR, 0.81; 0.70 to 0.92) was found in those cases of low mTG (<10th, 0.81 mM), but no correlation was found between low mTG levels and the risk of SGA. The results remained robust after excluding women with high or low body mass index (BMI) and pregnancy complications. CONCLUSIONS: This study suggested that early pregnancy exposure of mTG were related to the occurrence of SGA and LGA. mTG levels higher than 2.05 mM (>90th) were suggested to be avoid because of its risk for LGA, while mTG lower than 0.81 mM (<10th) showed its benefits for ideal birthweight range.

Six new species of zombie-ant fungi from Yunnan in China.

Some Ophiocordyceps species infecting ants are able to manipulate the host behavior. The hosts are manipulated in order to move to location that are advantageous for fungal spore transmission. Ophiocordyceps species that are able to manipulate the ant's behavior are called "zombie-ant fungi". They are widespread within tropical forests worldwide, with relatively few reports from subtropical monsoon evergreen broad-leaf forest. Zombie-ant fungi have been described and reported in different countries worldwide. However, there were a few reports from China. This study proposed six new species of zombie-ant fungi from China based on multi-gene (SSU, LSU, TEF, RPB1 and RPB2) phylogenetic analyses and morphological characteristics. Six novel species of Ophiocordyceps from China were identified as the Ophiocordyceps unilateralis core clade, forming a separate lineage with other species. Six novel species of Ophiocordyceps with hirsutella-like asexual morphs exclusively infecting ants were presented herein, namely, Ophiocordyceps acroasca, Ophiocordyceps bifertilis, Ophiocordyceps subtiliphialida, Ophiocordyceps basiasca, Ophiocordyceps nuozhaduensis and Ophiocordyceps contiispora. Descriptions and illustrations for six taxon were provided. Five of these species were collected from the subtropical monsoon evergreen broad-leaf forest, and one was collected from the rainforest and subtropical monsoon evergreen broad-leaf forest. This work proposes that the same host of Camponotus can be infected by multiple ant pathogenic fungi, while multiple ants of Polyrhachis can be infected by the same pathogenic fungi at the same time. This study contributes towards a better understanding of the evolutionary relationship between hosts and fungi, and provides novel insights into the morphology, distribution, parasitism, and ecology of Ophiocordyceps unilateralis sensu lato. We have provided a method for obtaining living cultures of Ophiocordyceps unilateralis complex species and their asexual morphs based on the living cultures, which is of significant value for further studies of Ophiocordyceps unilateralis complex species in the future.

A senescence-associated signature refines the classification of different modification patterns and characterization of tumor immune microenvironment infiltration in triple-negative breast cancer.

Background: Recent studies have found that senescence-associated genes play a significant role in cancer biological processes. We aimed to analyze the characteristics and role of senescence-associated genes in triple-negative breast cancer (TNBC). Methods: We systematically screened senescence-associated secretory phenotype (SASP) genes based on the gene expression information in the TCGA database. According to the expression levels of senescence-associated genes, TNBC was classified into two subtypes, namely, TNBCSASP1 and TNBCSASP2, using an unsupervised cluster algorithm. We then performed gene expression, enrichment pathway, immune infiltration, mutational profile characterization, drug sensitivity and prognostic value analyses for the two subtypes. The reliability and prognostic predictive utility of this classification model were validated. The most prognostically relevant gene, FAM3B, was comprehensively identified and validated by tissue microarray in TNBC. Results: TNBC was classified into two senescence-associated subtypes, TNBCSASP1 and TNBCSASP2, based on the set of senescence-associated secretory phenotype genes, among which the TNBCSASP1 subtype had a poor prognosis. The TNBCSASP1 subtype was immunosuppressed, with suppressed immune-related signaling pathways and low immune cell infiltration. The effect of the mutation on the TP53 and TGF-ß pathways could be related to the poor prognosis of the TNBCSASP1 subtype. Drug sensitivity analysis showed that AMG.706, CCT007093, and CHIR.99021 were potential targeted drugs for the TNBCSASP1 subtype. Finally, FAM3B was a key biomarker affecting the prognosis of patients with triple-negative breast cancer. Compared to normal breast tissue, the expression of FAM3B was reduced in triple-negative breast cancer. Survival analysis showed that overall survival was significantly shorter in triple-negative breast cancer patients with high FAM3B expression. Conclusion: A senescence-associated signature with different modification patterns has critical potential for providing a better understanding of TNBC biological processes, and FAM3B might serve as an applicable target for TNBC therapy.

Neoadjuvant docetaxel, epirubicin, and cyclophosphamide with or without metformin in breast cancer patients with metabolic abnormality: results from the randomized Phase II NeoMET trial.

PURPOSE: Breast cancer patients with metabolic syndrome (MetS) and its components show worse treatment responses to chemotherapy. Metformin is a widely used antidiabetic drug which also shows potential anticancer effect. This study aims to evaluate the efficacy, safety, and metabolic parameters change of metformin combined with docetaxel, epirubicin, and cyclophosphamide (TEC) in neoadjuvant treatment (NAT) for breast cancer patients with metabolic abnormality. METHODS: Eligible breast cancer patients were randomized to receive six cycles of TEC (docetaxel 75 mg/m2, epirubicin 75 mg/m2, and cyclophosphamide 500 mg/m2, d1, q3w) or TEC with metformin (TECM, TEC with oral metformin 850 mg once daily for the first cycle, then 850 mg twice daily for the following cycles). The primary end point was total pathological complete response (tpCR, ypTis/0N0) rate. RESULTS: Ninety-two patients were enrolled and randomized from October 2013 to December 2019: 88 patients were available for response and safety assessment. The tpCR rates were 12.5% (5/40) and 14.6% (7/48) in the TEC and TECM groups, respectively (P = 0.777). There was no difference in Ki67 decrease after NAT between two groups (P = 0.456). Toxicity profile were similar between two groups. No grade 3 or higher diarrhea were recorded. Total cholesterol (TC) and high-density lipoprotein cholesterol worsened after NAT in the TEC arm but remained stable in the TECM arm. The absolute increase of TC and low-density lipoprotein cholesterol (LDL-C) was significantly lower in the TECM group compared with the TEC group. After a median follow-up of 40.8 (4.7-70.8) months, no survival difference was observed between TEC and TECM groups (all P > 0.05). CONCLUSION: Adding metformin to TEC didn't increase pCR rate and disease outcome in breast cancer patients with metabolic abnormality. However, additional metformin treatment with chemotherapy would prevent TC and LDL-C increase after NAT. Trial Registration ClinicalTrials.gov Identifier: NCT01929811.

N-terminal propeptide of type 3 collagen-based sequential algorithm can identify high-risk steatohepatitis and fibrosis in MAFLD.

BACKGROUND AND AIMS: With metabolic dysfunction-associated fatty liver disease (MAFLD) incidence and prevalence sharply increasing globally, there is an urgent need for non-invasive diagnostic tests to accurately screen high-risk MAFLD patients for liver inflammation and fibrosis. We aimed to develop a novel sequential algorithm based on N-terminal propeptide of type 3 collagen (PRO-C3) for disease risk stratification in patients with MAFLD. METHODS: A derivation and independent validation cohort of 327 and 142 patients with biopsy-confirmed MAFLD were studied. We compared the diagnostic performances of various non-invasive scores in different disease states, and a novel sequential algorithm was constructed by combining the best performing non-invasive scores. RESULTS: For patients with high-risk progressive steatohepatitis (i.e., steatohepatitis + NAFLD activity score ≥ 4 + F ≥ 2), the AUROC of FAST score was 0.801 (95% confidence interval (CI): 0.739-0.863), and the negative predictive value (NPV) was 0.951. For advanced fibrosis (≥ F3) and cirrhosis (F4), the AUROCs of ADAPT and Agile 4 were 0.879 (95%CI 0.825-0.933) and 0.943 (95%CI 0.892-0.994), and the NPV were 0.972 and 0.992. Sequential algorithm of ADAPT + Agile 4 combination was better than other combinations for risk stratification of patients with severe fibrosis (AUROC = 0.88), with similar results in the validation cohort. Meanwhile, in all subgroup analyses (stratifying by sex, age, diabetes, NAS, BMI and ALT), ADAPT + Agile 4 had a good diagnostic performance. CONCLUSIONS: The new sequential algorithm reliably identifies liver inflammation and fibrosis in MAFLD, making it easier to exclude low-risk patients and recommending high-risk MAFLD patients for clinical trials and emerging pharmacotherapies.

Fully connected neural network-based serum surface-enhanced Raman spectroscopy accurately identifies non-alcoholic steatohepatitis.

BACKGROUND/PURPOSE OF THE STUDY: There is a need to find a standardized and low-risk diagnostic tool that can non-invasively detect non-alcoholic steatohepatitis (NASH). Surface enhanced Raman spectroscopy (SERS), which is a technique combining Raman spectroscopy (RS) with nanotechnology, has recently received considerable attention due to its potential for improving medical diagnostics. We aimed to investigate combining SERS and neural network approaches, using a liver biopsy dataset to develop and validate a new diagnostic model for non-invasively identifying NASH. METHODS: Silver nanoparticles as the SERS-active nanostructures were mixed with blood serum to enhance the Raman scattering signals. The spectral data set was used to train the NASH classification model by a neural network primarily consisting of a fully connected residual module. RESULTS: Data on 261 Chinese individuals with biopsy-proven NAFLD were included and a prediction model for NASH was built based on SERS spectra and neural network approaches. The model yielded an AUROC of 0.83 (95% confidence interval [CI] 0.70-0.92) in the validation set, which was better than AUROCs of both serum CK-18-M30 levels (AUROC 0.63, 95% CI 0.48-0.76, p = 0.044) and the HAIR score (AUROC 0.65, 95% CI 0.51-0.77, p = 0.040). Subgroup analyses showed that the model performed well in different patient subgroups. CONCLUSIONS: Fully connected neural network-based serum SERS analysis is a rapid and practical tool for the non-invasive identification of NASH. The online calculator website for the estimated risk of NASH is freely available to healthcare providers and researchers ( http://www.pan-chess.cn/calculator/RAMAN_score ).

Genomic Comparative Analysis of Cordyceps pseudotenuipes with Other Species from Cordyceps.

The whole genome of Cordyceps pseudotenuipes was sequenced, annotated, and compared with three related species to characterize the genome. The antibiotics and Secondary Metabolites Analysis Shell (antiSMASH) and local BLAST analysis were used to explore the secondary metabolites (SMs) and biosynthesis gene clusters (BGCs) of the genus Cordyceps. The genome-wide basic characteristics of C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris revealed unequal genome size, with C. cicadae as the largest (34.11 Mb), followed by C. militaris (32.27 Mb). However, the total gene lengths of C. pseudotenuipes and C. tenuipes were similar (30.1 Mb and 30.06 Mb). The GC contents of C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris genomes differed slightly (51.40% to 54.11%). AntiSMASH and local BLAST analysis showed that C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris had 31, 28, 31, and 29 putative SM BGCs, respectively. The SM BGCs contained different quantities of polyketide synthetase (PKS), nonribosomal peptide synthetase (NRPS), terpene, hybrid PKS + NRPS, and hybrid NRPS + Other. Moreover, C. pseudotenuipes, C. tenuipes, C. cicadae, and C. militaris had BGCs for the synthesis of dimethylcoprogen. C. pseudotenuipes, C. tenuipes, and C. cicadae had BGCs for the synthesis of leucinostatin A/B, neosartorin, dimethylcoprogen, wortmanamide A/B, and beauvericin. In addition, the SM BGCs unique to C. pseudotenuipes were clavaric acid, communesin, and deoxynivalenol. Synteny analysis indicated that the scaffolds where the SM BGC was located were divided into more than 70 collinear blocks, and there might be rearrangements. Altogether, these findings improved our understanding of the molecular biology of the genus Cordyceps and will facilitate the discovery of new biologically active SMs from the genus Cordyceps using heterologous expression and gene knockdown methods.

Association of Molecular Biomarker Heterogeneity With Treatment Pattern and Disease Outcomes in Multifocal or Multicentric Breast Cancer.

Purpose: This study aimed to evaluate the rates of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 heterogeneity in multifocal or multicentric breast cancer (MMBC) and its association with treatment pattern and disease outcomes. Methods: MMBC patients with ER, PR, HER2, and Ki67 results for each tumor focus were retrospectively analyzed using Kappa test and categorized into the homogeneous group (Homo group) and the heterogeneous group (Hetero group). Chi-square tests were performed to compare the clinical features and treatment options between the groups. Disease-free survival (DFS) and overall survival (OS) rates were estimated from Kaplan-Meier curves and compared between two groups. Results: A total of 387 patients were included, and 93 (24.0%) were classified into the Hetero group. Adjuvant endocrine therapy was more frequently assigned for patients in the Hetero group than in the Homo group (84.9% vs. 71.7%, p = 0.046). There was no difference in terms of adjuvant anti-HER2 therapy (28.3% vs. 19.6%, p = 0.196) and chemotherapy (69.9% vs. 69.8%, p = 0.987) usage between the two groups. At a median follow-up of 36 months, DFS rates were 81.2% for the Hetero group and 96.5% for the Homo group (p = 0.041; adjusted HR, 2.95; 95% CI, 1.04-8.37). The estimated 3-year OS rates for the groups were 95.8% and 99.5%, respectively (p = 0.059; adjusted HR, 5.36; 95% CI, 0.97-29.69). Conclusion: Heterogeneity of ER, PR, HER2, or Ki67 was present in 24.0% patients with MMBC. Biomarkers heterogeneity influenced adjuvant endocrine therapy usage and was associated with worse disease outcomes, indicating further clinical evaluation.

The Comparative Safety of Epirubicin and Cyclophosphamide versus Docetaxel and Cyclophosphamide in Lymph Node-Negative, HR-Positive, HER2-Negative Breast Cancer (ELEGANT): A Randomized Trial.

Background: In adjuvant settings, epirubicin and cyclophosphamide (EC) and docetaxel and cyclophosphamide (TC) are both optional chemotherapy regimens for lymph node-negative, hormone receptor (HR)-positive, human epidermal receptor 2 (HER2)-negative breast cancer patients. Neutropenia is one of the most common adverse events (AEs) of these regimens. The rate of grade 3−4 neutropenia varies in different studies, and direct comparisons of safety profiles between EC and TC are lacking. Method: ELEGANT (NCT02549677) is a prospective, randomized, open-label, noninferior hematological safety trial. Eligible patients with lymph node-negative HR+/HER2-tumors (1:1) were randomly assigned to received four cycles of EC (90/600 mg/m2) or TC (75/600 mg/m2) every three weeks as adjuvant chemotherapy. The primary endpoint was the incidence of grade 3 or 4 neutropenia defined by National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 on an intention-to-treat basis. Noninferiority was defined as an upper 95% CI less than a noninferiority margin of 15%. Results: In the intention-to-treat population, 140 and 135 patients were randomized into the EC and TC arms, respectively. For the primary endpoint, the rate of grade 3 or 4 neutropenia is 50.71% (95% CI: 42.18%, 59.21%) in the EC arm and 48.15% (95% CI: 39.53%, 56.87%) in the TC arm (95%CI risk difference: −0.100, 0.151), showing the noninferiority of the EC arm. For secondary endpoints, the rate of all-grade anemia is higher in the EC arm (EC 42.86% versus TC 22.96%, p = 0.0007), and more patients suffer from nausea/vomiting, hair loss, and nail changes (p < 0.01) in the EC arm. No statistically different disease-free survival was observed between the two arms (p = 0.13). Conclusion: EC is not inferior to TC in the rate of grade 3 or 4 neutropenia, but more other AEs were observed in the EC group.

PNPLA3 rs738409 C>G Variant Influences the Association Between Visceral Fat and Significant Fibrosis in Biopsy-proven Nonalcoholic Fatty Liver Disease.

Background and Aims: Intra-abdominal visceral fat accumulation and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 G/C gene polymorphism confer a greater susceptibility to nonalcoholic fatty liver disease (NAFLD). We examined whether the relationship between visceral fat accumulation and liver disease severity may be influenced by PNPLA3 rs738409 polymorphism. Methods: The variant of PNPLA3 rs738409 was genotyped within 523 Han individuals with biopsy-confirmed NAFLD. Visceral fat area (VFA) was measured by bioelectrical impedance. Significant liver fibrosis (SF), defined as stage F ≥2 on histology, was the outcome measure of interest. Results: The distribution of PNPLA3 genotypes was CC: 27.5%, CG: 48.2%, and GG: 24.3%. Higher VFA was associated with greater risk of having SF (adjusted-odds ratio [OR]: 1.03; 95% confidence interval [CI]: 1.02-1.04, p<0.05), independent of potential confounders. Among subjects with the same VFA level, the risk of SF was greater among carriers of the rs738409 G genotype than among those who did not. Stratified analysis showed that PNPLA3 rs738409 significantly influenced the association between VFA and SF. VFA remained significantly associated with SF only among the rs738409 G-allele carriers (adjusted-OR: 1.05; 95% CI: 1.03-1.08 for the GG group; and adjusted-OR:1.03; 95% CI: 1.01-1.04 for the GC group). There was a significant interaction between VFA and PNPLA3 rs738409 genotype (P interaction =0.004). Conclusions: PNPLA3 rs738409 G allele has a moderate effect on the association between VFA and risk of SF in adult individuals with biopsy-proven NAFLD. Existence of the PNPLA3 rs738409 G allele and VFA interact to increase risk of SF.

Low skeletal muscle mass is associated with more severe histological features of non-alcoholic fatty liver disease in male.

BACKGROUND/PURPOSE OF THE STUDY: Although low skeletal muscle mass is associated with non-alcoholic fatty liver disease (NAFLD), it is currently uncertain whether there are associations between weight-adjusted appendicular skeletal muscle (ASM%), severity of histological features of NAFLD, and the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism. Our aim was to test for a possible influence of the PNPLA3 rs738409 variant on the association between ASM% and severity of NAFLD histological features. METHODS: We enrolled 401 Chinese male with biopsy-proven NAFLD. Using a bioelectrical-impedance body composition analyzer (BIA, Inbody 720, Japan Inc., Tokyo), we calculated the ASM% as the percentage of total appendicular skeletal muscle mass (ASM, kg)/total body mass (kg) × 100. RESULTS: Compared to those with high ASM%, patients with low ASM% (≤ 30.6, i.e., the median value of distribution of the whole sample) had a greater severity of individual histological features of NAFLD. These patients also had a higher risk of severe steatosis and non-alcoholic steatohepatitis (NASH) (adjusted-odds ratio [OR] 2.34, 95% CI 1.39-3.93, and adjusted-OR 2.22, 95% CI 1.30-3.77) even after adjusting for age, body mass index, diabetes, and serum creatinine levels. Carriage of the G allele of PNPLA3 rs738409 plus low ASM% was associated with a higher risk of severe steatosis and presence of liver fibrosis (OR 3.02, 95% CI 1.46-6.26, p = 0.003 and OR 2.18, 95% CI 1.03-4.60, p = 0.041 respectively), and there was a non-significant but borderline increased risk of NASH (OR 2.00, 95% CI 0.98-4.06, p = 0.056). CONCLUSIONS: Low ASM% and the presence of a G allele within PNPLA3 rs738409 is associated with more severe histological features of NAFLD.