Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation.

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Longitudinal ultrasound-based AI model predicts axillary lymph node response to neoadjuvant chemotherapy in breast cancer: a multicenter study.

OBJECTIVES: Developing a deep learning radiomics model from longitudinal breast ultrasound and sonographer's axillary ultrasound diagnosis for predicting axillary lymph node (ALN) response to neoadjuvant chemotherapy (NAC) in breast cancer. METHODS: Breast cancer patients undergoing NAC followed by surgery were recruited from three centers between November 2016 and December 2022. We collected ultrasound images for extracting tumor-derived radiomics and deep learning features, selecting quantitative features through various methods. Two machine learning models based on random forest were developed using pre-NAC and post-NAC features. A support vector machine integrated these data into a fusion model, evaluated via the area under the curve (AUC), decision curve analysis, and calibration curves. We compared the fusion model's performance against sonographer's diagnosis from pre-NAC and post-NAC axillary ultrasonography, referencing histological outcomes from sentinel lymph node biopsy or axillary lymph node dissection. RESULTS: In the validation cohort, the fusion model outperformed both pre-NAC (AUC: 0.899 vs. 0.786, p < 0.001) and post-NAC models (AUC: 0.899 vs. 0.853, p = 0.014), as well as the sonographer's diagnosis of ALN status on pre-NAC and post-NAC axillary ultrasonography (AUC: 0.899 vs. 0.719, p < 0.001). Decision curve analysis revealed patient benefits from the fusion model across threshold probabilities from 0.02 to 0.98. The model also enhanced sonographer's diagnostic ability, increasing accuracy from 71.9% to 79.2%. CONCLUSION: The deep learning radiomics model accurately predicted the ALN response to NAC in breast cancer. Furthermore, the model will assist sonographers to improve their diagnostic ability on ALN status before surgery. CLINICAL RELEVANCE STATEMENT: Our AI model based on pre- and post-neoadjuvant chemotherapy ultrasound can accurately predict axillary lymph node metastasis and assist sonographer's axillary diagnosis. KEY POINTS: Axillary lymph node metastasis status affects the choice of surgical treatment, and currently relies on subjective ultrasound. Our AI model outperformed sonographer's visual diagnosis on axillary ultrasound. Our deep learning radiomics model can improve sonographers' diagnosis and might assist in surgical decision-making.

Fecal microbial biomarkers in older adults with autoimmune diseases.

Aim: To analyze the alterations in the fecal microbiota of older adults with autoimmune disease and determine the diagnostic capabilities of microbial biomarkers. Methods: The raw data of fecal samples from 444 older adults from the publicly available American Gut Project database was analyzed. Results: It was found that there were no significant differences in the microbiota richness and evenness between older adults with autoimmune disease and healthy controls. However, significant differences were observed in the microbiota composition and structure. The subject operating characteristic curve of the eight key microbiota was obtained, and the area under curve value was 70.0%. Conclusion: Older adults with autoimmune disease showed changes in intestinal microbiota composition, which can be used as microbial biomarkers.

Bone Marrow Histology in Hemophagocytic Lymphohistiocytosis.

CONTEXT.­: Bone marrow (BM) samples are obtained through aspiration and trephine biopsy. Hemophagocytic lymphohistiocytosis (HLH) has been largely studied in BM aspirate smears. OBJECTIVE.­: To investigate the histologic features of HLH in trephine biopsy. DESIGN.­: Patients with hemophagocytosis in BM aspirate smears were assigned to HLH (n = 127) and non-HLH (n = 203) groups. We quantified hematoxylin-eosin and CD68 immunohistochemical staining of their trephine biopsies. RESULTS.­: No significant correlation was noted in the hemophagocytosis count between aspirate smears and trephine biopsies. Compared with the non-HLH group, the HLH group had a higher hemophagocytosis count (13 versus 9 per tissue section, P = .046), lower percentage of the adipocytic area (36.7% versus 50.3%, P < .001), and higher percentage of the foamy area (19.1% versus 14.5%, P < .001). The HLH group had more histiocyte infiltrates (total histiocyte density, 9.2% versus 7.3%; P < .001) and more fat-infiltrating histiocytes (histiocyte density of the fat-associated part [HD-FA], 7.6% versus 6.2%; P < .001). We identified the following poor prognostic factors in the HLH group: age 50 years or older (median overall survival [mOS], 95 versus 499 days; P = .04), Epstein-Barr virus-positive T-cell lymphoproliferative diseases (EBV+TLPDs) (mOS, 51 versus 425 days; P < .001), hemophagocytosis count of 6 or higher per tissue section (mOS, 66 versus 435 days; P = .02), and HD-FA of 9% or greater (mOS, 61 versus 359 days; P = .02). Multivariate analysis revealed that age 50 years or older (hazard ratio [HR], 2.38; P < .001), EBV+TLPDs (HR, 2.07; P < .001), and hemophagocytosis count of 6 or higher per tissue section (HR, 2.07; P = .002) were independent prognostic factors for HLH. CONCLUSIONS.­: The HLH group had higher hemophagocytic activity, higher cellularity, a more foamy appearance, more histiocyte infiltrates, and more fat-infiltrating histiocytes. High hemophagocytic activity and marked histiocyte infiltrates in the BM fat were associated with poorer prognosis.

Roles of oral microbiota and oral-gut microbial transmission in hypertension.

INTRODUCTION: Considerable evidence has linked periodontitis (PD) to hypertension (HTN), but the nature behind this connection is unclear. Dysbiosis of oral microbiota leading to PD is known to aggravate different systematic diseases, but the alteration of oral microbiota in HTN and their impacts on blood pressure (BP) remains to be discovered. OBJECTIVES: To characterize the alterations of oral and gut microbiota and their roles in HTN. METHODS: We performed a cross-sectional (95 HTN participants and 39 controls) and a 6-month follow-up study (52 HTN participants and 26 controls) to analyze the roles of oral and gut microbiota in HTN. Saliva, subgingival plaques, and feces were collected for 16S rRNA gene sequencing or metagenomic analysis. C57BL/6J mice were pretreated with antibiotics to deplete gut microbiota, and then transplanted with human saliva by gavage to test the impacts of abnormal oral-gut microbial transmission on HTN. RESULTS: BP in participants with PD was higher than no PD in both cross-sectional and follow-up cohort. Relative abundances of 14 salivary genera, 15 subgingival genera and 10 gut genera significantly altered in HTN and those of 7 salivary genera, 12 subgingival genera and 6 gut genera significantly correlated with BP. Sixteen species under 5 genera were identified as oral-gut transmitters, illustrating the presence of oral-gut microbial transmission in HTN. Veillonella was a frequent oral-gut transmitter stably enriched in HTN participants of both cross-sectional and follow-up cohorts. Saliva from HTN participants increased BP in hypertensive mice. Human saliva-derived Veillonella successfully colonized in mouse gut, more abundantly under HTN condition. CONCLUSIONS: PD and oral microbiota are strongly associated with HTN, likely through oral-gut transmission of microbes. Ectopic colonization of saliva-derived Veillonella in the gut may aggravate HTN. Therefore, precise manipulations of oral microbiota and/or oral-gut microbial transmission may be useful strategies for better prevention and treatment of HTN.

Development of peroxynitrite-responsive fluorescence probe for recognition of drug-induced liver injury.

Peroxynitrite (ONOO-) as an active substance, is produced during normal physiological process, which plays an important role in maintaining cell REDOX balance and cell function. Moreover, the peroxynitrite is involved in many diseases and especially can be used as a biomarker of drug-induced liver injury (DILI). Therefore, in this work, we synthesized a fluorescent probe JQ-3 for detecting ONOO-. The results showed the probe JQ-3 possessed excellent selectivity, fast response time (10 min) and low detection limit (32 nM). The probe JQ-3 is almost unaffected by pH, showing the potential application in biological systems. Moreover, the probe JQ-3 can be successfully used for the detection of exogenous and endogenous ONOO- in living cells and zebrafish. At the same time, the DILI was successfully recognized by visualizing ONOO- with JQ-3 in living cells and zebrafish. Therefore, the probe JQ-3 provides a potential tool for detecting ONOO- to understand physiological and pathology processes of disease.

A highly selective probe for ratiometric imaging peroxynitrite in living cells and in vivo.

In this study, we constructed and displayed a ratiometric fluorescent probe JQ-2 for detecting ONOO-. The probe JQ-2 showed a ratiometric signal for visualizing ONOO- with a rapid response and high selectivity over a panel of biological analytes. Moreover, the JQ-2 has near-infrared emission (657 nm), which provides an excellent basis for the practical application in biological systems. The probe JQ-2 possessed low cytotoxicity and excellent cell membrane permeability, which can specifically visualize the exogenous and endogenous ONOO- in vitro and vivo by emission in two channels. Meanwhile, JQ-2 can be used for diagnosing drug-induced liver injury by visualizing and monitoring the fluctuations of endogenous ONOO-. Therefore, JQ-2 provided a potential tool for precisely detecting the fluctuation of ONOO- in biological systems to understand physiological and pathological process.

Diffuse midline glioma with H3-K27M mutation: A rare case with GFAP-positive anucleate whorled patterns.

INTRODUCTION: Diffuse midline glioma with H3-K27M mutation is an infiltrative high-grade glioma, with predominantly astrocytic differentiation. PATIENT CONCERNS: A 54-year-old Chinese woman presented with memory loss for a month and walking instability for 15 days. DIAGNOSIS: Magnetic resonance imaging showed a mass shadow of isometric T1 and slightly longer T2 with mild mixed signals in the third ventricle of the suprasellar region. Histologically, the tumor was primarily sheet-like, with many "anucleate areas" composed of long and thin fibrillary processes of the bipolar cells, which formed "whorls." The neoplastic nuclei were ovoid and moderate in size. The tumor showed brisk mitotic activity and vascular proliferation, with no necrosis. In addition to histone H3K27M mutation, immunohistochemical staining showed that the tumor cells were positive for glial fibrillary acidic protein, oligodendrocyte transcription factor 2, alpha-thalassemia/mental retardation syndrome X, S-100 and Vimentin. The "anucleate areas" were positive for glial fibrillary acidic protein and negative for synaptophysin. The Ki-67 proliferation index was about 10%. Molecular genetic analyses detected H3F3A K27M mutation, but no mutations in IDH1 or IDH2, TERT promoter mutations, MGMT promoter methylation, KIAA1549-BRAF fusion or deletion of 1p/19q were found. Based on these findings, the patient was diagnosed as diffuse midline glioma with H3-K27M mutation in the third ventricle, corresponding to WHO grade 4. INTERVENTIONS: A craniotomy with total excision of the tumor was performed. OUTCOMES: After surgery, she was routinely treated with temozolomide for chemotherapy and synchronous radiotherapy. It has been 11 months now, and the patient is living well. CONCLUSION: This case report provides information on the microscopic morphological features of diffuse midline glioma with H3K27M mutation, which can help pathologists to make a definitive diagnosis of this tumor.

Radiologic and Pathological Investigation of Pseudarthrosis in Ankylosing Spondylitis: Distinguishing Between Inflammatory and Traumatic Etiology.

OBJECTIVE: To investigate the pathogenesis of pseudarthrosis in ankylosing spondylitis (AS) based on the pathological analysis of specimens harvested during surgery. METHODS: Radiographic and clinical data for 17 consecutive AS patients with pseudarthrosis were retrospectively analyzed. Meanwhile, the pathological analysis of specimens obtained during surgery was also performed. RESULTS: In total, 18 extensive Andersson lesions were included. Pseudarthrosis located at the apical region were noted in 12 patients. Complete ossified anterior longitudinal ligaments above or below pseudarthrosis and fracture through posterior elements or facet joints were observed in 7 and 6 lesions, respectively. The most definitive pathological characteristic in all cases was proliferating hypovascular edematous fibrous tissue involving disc, bone-disc border, and vertebral body. Fibrinoid necrosis, necrotic bone fragments, hemosiderin deposits, and active subchondral osteogenesis were found, indicating trauma process. Mild perivascular collections of inflammatory cells were detected in only 2 cases. CONCLUSION: AS-related pseudarthrosis is more likely to originate from mechanical trauma than inflammation. The above-mentioned radiological and histological findings showed that multiple mechanisms lead to the formation of pseudarthrosis. These mechanisms include excessive stress, insufficiency fracture, and an acute fracture involving a 3-column structure.

A Catechol-Type Resveratrol Analog Manifests Antiangiogenic Action by Constructing an Efficient Catalytic Redox Cycle with Intracellular Copper Ions and NQO1.

SCOPE: As part of our research project to understand why dietary polyphenols with the catechol skeleton tend to exhibit cancer chemopreventive activity, a catechol-type resveratrol analog (3,4-dihydroxy-trans-stilbene [3,4-DHS]) was selected to probe its antiangiogenic effects and mechanisms. METHODS AND RESULTS: The antiangiogenic effects of 3,4-DHS on angiogenesis-related endothelial cell functions were examined, including migration, invasion, and tube formation, and in vivo angiogenesis on a chick chorioallantoic membrane assay. The potential molecular mechanisms for the suppression of cell migration by 3,4-DHS were analyzed using various specific inhibitors. 3,4-DHS was identified as a potent angiogenesis inhibitor by constructing an efficient catalytic redox cycle with intracellular copper ions and NAD(P)H quinone oxidoreductase I to generate reactive oxygen species and thereby downregulate matrix metalloproteinase-9. CONCLUSION: This work provides further evidence that dietary catechols manifest antiangiogenic activity by virtue of their copper-dependent prooxidative instead of antioxidative role, and useful information for designing polyphenol-inspired angiogenesis inhibitors.

Regional and seasonal effects on the gastrointestinal parasitism of captive forest musk deer.

Parasite infections can cause adverse effects on health, survival and welfare of forest musk deer. However, few studies have quantified the parasite infection status and evaluated the parasite temporal dynamics and differences between breeding centers for captive forest musk deer. The purpose of this study was to assess seasonal and regional effects on the parasite prevalence, shedding capacity, diversity, aggregation and infracommunity to establish baseline data on captive forest musk deer. The McMaster technique was applied to count parasite eggs or oocysts in 990 fecal samples collected at three breeding centers located in Qinling Mountains and Tibetan Plateau during spring, summer, and winter. Five gastrointestinal parasite groups were found in musk deer, and Eimeria spp. were dominant (mean oocysts per gram=1273.7±256.3). A positive correlation between Eimeria spp. and Strongyloides spp. (r=0.336, p<0.001) based on shedding capacity data was found, as well as a negative correlation between Eimeria spp. and Moniezia spp. (r=-0.375, p=0.003). Both seasonal and regional differences in diversity, prevalence, shedding capacity, aggregation and infracommunity were observed for five parasite groups. The low level of aggregation and high shedding capacity of Eimeria spp. and Strongyloides spp. might reflect the contaminated environment, and indicate that host-parasite relationships are unstable. The high degree of aggregation of Trichuris spp., Ascaris spp., and Moniezia spp. also suggests that some individual hosts had less ability to resist pathogens and greater transmission potential than others. These conclusions suggest that a focus on disease control strategies could improve the health of forest musk deer in captivity.

Follow-up Results and Clinical Significance of Magnetic Resonance Spectroscopy Combined with Fluorescence Microscopy in Resection of High Grade Supra-tentoria Gliomas.

Objective To investigate the clinical effectiveness of magnetic resonance spectroscopy (MRS) combined with sodium fluorescein(FL) in the treatment of high grade gliomas(HGG). Methods From August 2013 to 2015 November,the clinical data of 72 supratentorial HGG(WHO grade Ⅲ-Ⅳ) patients who had received surgical treatment in our hospital were retrospectively studied,among whom 43 cases received MRS combined with intra-perative FL navigation(observation group),and 29 cases only received conventional surgery(control group). Post-operative radiotherapy and chemotherapy were applied for more than 3 months. Routine enhanced MRI were performed 24-48 hours after the operation to investigate the extent of tumor resection. Six months after the operation,the quality of life of patients was evaluated by using the Karnofsky score,and 1-year postoperative survival rate and progression-free survival(PFS) were observed. Results Postoperative MRI showed that the rate of gross total resection(GTR) in observation group was significantly higher than that in control group(72.09%vs.51.72%;χ2=23.88,P=0.001),and the GTR rate of WHO grade Ⅳ tumors was significantly higher than that of WHO grade Ⅲ tumors in observation group(92.86% vs.62.07%;χ2=6.06,P=0.042). The postoperative Karnofsky score in the observation group was significantly higher than that in control group(µ=2.34,P=0.021). The mean time of follow-up was(16.4±2.4) months(8-21 months) and there was no statistical significant difference between observation group and control group in 1-year survival rate(74.07% vs.77.50%;χ2=4.90,P=0.165) and PFS [(13.2±1.2) months vs.(12.7±2.0) months;χ2=7.26,P=0.067]. In observation group,the PFS of WHO grade Ⅳ patients was significantly higher than that in control group [(14.2±0.3) months vs.(10.0±1.1) months;χ2=11.03,P=0.031]. There was also no statistical significant difference between WHO grade Ⅳ tumors in two groups in terms of 1-year survival rate(71.43% vs.72.54%;χ2=5.33,P=0.089),and there was no statistical significant difference between WHO grade Ⅲ tumors in two groups in 1-year survival rate(75.86% vs. 72.22%;χ2=3.78,P=0.250) and in PFS [(13.7±1.4) months vs.(12.4±0.8) months;χ2=4.85,P=0.083]. Conclusions MRS combined with intraoperative FL navigation technology can improve the resection rate and improve survival quality of patients,and there is no evidence that MRS combined with intraoperative FL navigation prolong the overall survival of patients with high-grade gliomas. Different outcome may be found with longer follow-up and increased simple size.

Primary Cutaneous Sarcomatoid Anaplastic Lymphoma Kinase-Positive Anaplastic Large-Cell Lymphoma With Linear Distributional Lesions.

We report a case of cutaneous anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALCL) with linear distributional lesions and sarcomatoid histologic features. A sarcomatoid variant is the rarest morphological pattern of ALCL. Interestingly, the morphology of tumor cells in the present case transitioned from a sarcomatoid variant of ALCL at first diagnosis to a classic variant at relapse. The case is a diagnostic challenge considering both the clinical and histologic aspects. Awareness of the sarcomatoid variant of ALCL and its morphological changes can lead to a correct diagnosis.

Lethal albinic seedling, encoding a threonyl-tRNA synthetase, is involved in development of plastid protein synthesis system in rice.

KEY MESSAGE: An albinic rice is caused by mutation of threonyl-tRNA synthetase, which is essential for plant development by stabilizing of NEP and PEP gene expressions and chloroplast protein synthesis. Chloroplast biogenesis and development depend on complex genetic mechanisms. Apart from their function in translation, aminoacyl-tRNA synthetases (aaRSs) play additional role in gene expression regulation, RNA splicing, and cytokine activity. However, their detailed functions in plant development are still poorly understood. We isolated a lethal albinic seedling (las) mutant in rice. Physiological and ultrastructural analysis of las mutant plants revealed weak chlorophyll fluorescence, negligible chlorophyll accumulation, and defective thylakoid membrane development. By map based cloning we determined that the LAS allele gene encodes threonyl-tRNA synthetase (ThrRS). LAS was constitutively expressed with relatively high level in leaves. NEP-dependent gene transcripts accumulated in the developing chloroplasts, while PEP-dependent transcripts were reduced in the las mutant. This result indicated that PEP activity was impaired. Chloroplast-encoded protein levels were sharply reduced in the las mutant. Biogenesis of chloroplast rRNAs (16S and 23S rRNA) was arrested, leading to impaired translation and protein synthesis. Together, our findings indicated that LAS is essential not only for chloroplast development by stabilizing the NEP and PEP gene expression, but also for protein synthesis and construction of the ribosome system in rice chloroplasts.

The Thr300Ala variant of ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) often metastasizes to the brain, but identifying which patients will develop brain metastases (BM) is difficult. Macroautophagy/autophagy is critical for cancer initiation and progression. We hypothesized that genetic variants of autophagy-related genes may affect brain metastases (BM) in NSCLC patients. We genotyped 16 single nucleotide polymorphisms (SNPs) in 7 autophagy-related (ATG) genes (ATG3, ATG5, ATG7, ATG10, ATG12, ATG16L1, and MAP1LC3/LC3) by using DNA from blood samples of 323 NSCLC patients. Further, we evaluated the potential associations of these genes with subsequent BM development. Lung cancer cell lines stably transfected with ATG16L1: rs2241880 (T300A) were established. Mouse models of brain metastasis were developed using cells transfected with ATG16L1-300T or ATG16L1-300A. ATG10: rs10036653 and ATG16L1: rs2241880 were significantly associated with a decreased risk of BM (respective hazard ratios [HRs]=0.596, 95% confidence interval [CI] 0.398-0.894, P = 0.012; and HR = 0. 655, 95% CI 0.438-0.978, P = 0.039, respectively). ATG12: rs26532 was significantly associated with an increased risk of BM (HR=1.644, 95% CI 1.049-2.576, P = 0.030). Invasion and migration assays indicated that transfection with ATG16L1-300T (vs. 300A) stimulated the migration of A549 cells. An in vivo metastasis assay revealed that transfection with ATG16L1-300T (vs. 300A) significantly increased brain metastasis. Our results indicate that genetic variations in autophagy-related genes can predict BM and that genome analysis would facilitate stratification of patients for BM prevention trials.

Risk factors for the recurrence of an intracranial saccular aneurysm following endovascular treatment.

BACKGROUND: This study was aimed to determine risk factors for the recurrence of an intracranial saccular aneurysm (ISA) following endovascular treatment. The findings will help medical professionals to identify patients having a high risk of ISA recurrence and assist them in developing appropriate follow-up plans. RESULTS: During the follow-up period, 12.6% of the patients (83/658) experienced recurrent ISAs. An analysis of related factors, including gender, age, hypertension, diabetes mellitus, smoking, tumor size, width of the aneurysm neck, the presence or absence of a rupture, the volume embolization ratio (VER), the application or nonapplication of a stent, and follow-up time, revealed that a tumor size > 10 mm in diameter, wide-necked aneurysms, an anterior communicating or middle cerebral artery aneurysm, an aneurysm rupture, a VER < 20%, the absence of stent assistance, and follow-up time were high-risk factors for the recurrence of ISAs. MATERIALS AND METHODS: We retrospectively reviewed the records of 658 patients who underwent endovascular treatment for ISAs from January 2010 through December 2014. Multivariable logistic regression was performed on the candidates' risk factors, which were identified via univariable screening analysis. CONCLUSIONS: Smoking, a large tumor size, a wide-necked aneurysm, an anterior communicating or middle cerebral artery aneurysm, an aneurysm rupture, a VER < 20%, and an absence of stent assistance are significant risk factors for the postoperative recurrence of an aneurysm. Strict follow-up plans should be created for ISA patients having these high-risk factors.

Determination of ovarian cyclicity and pregnancy using fecal progesterone in forest musk deer (Moschus berezovskii).

The forest musk deer (FMD, Moschus berezovskii) is a threatened species in China. Although crucial for its artificial breeding management and thus protection, to date, gonadal steroidogenic activity data are unavailable in this species. Thus, the objectives of the present study were to characterize ovarian activity throughout the estrous cycle, non-pregnant luteal phase, and gestation in female FMD. These goals were accomplished using an enzyme immunoassay to measure fecal concentrations of estradiol (E2) and progesterone. Fecal samples were collected from female FMD (aged 3-4 years) for one year, including during breeding and non-breeding seasons. Non-pregnant estrous cycles were recorded in females, based on fecal progesterone concentrations, their overall estrous cycle length was (mean±SEM) 24.3±0.5 days, with 21.6±0.5 days in the luteal phase and 2.7±0.3 days in the inter-luteal phase. Fecal progesterone and E2 concentrations were also measured in females that became pregnant and gave birth after gestating approximately 6 months. Two weeks after becoming pregnant, the average fecal progesterone concentration was significantly greater than that during non-pregnancy. The average fecal progesterone concentrations during pregnancy increased 3.2-fold above non-pregnant concentrations, decreasing to non-pregnant concentrations only after parturition. By contrast, average fecal E2 concentrations during gestation and after parturition were not different from average non-pregnant concentration. In addition, no difference was observed between progesterone concentration in the first month after pregnancy and the value during the luteal phase. However, progesterone concentration during the second month of pregnancy was significantly higher than the value during the luteal phase. In conclusion, monitoring fecal progesterone is an effective method for assessing ovarian function in FMD and will be a useful tool for breeding management and development of assisted reproductive techniques, such as artificial insemination, in this species.

Development and mechanism investigation of a new piperlongumine derivative as a potent anti-inflammatory agent.

Inflammation, especially chronic inflammation, is directly involvement in the pathogenesis of many diseases including cancer. An effective approach for managing inflammation is to employ chemicals to block activation of nuclear factor-κB (NF-κB), a key regulator for inflammatory processes. Piperlongumine (piplartine, PL), an electrophilic molecule isolated from Piper longum L., possesses excellent anti-cancer and anti-inflammatory properties. In this study, a new PL analogue (PL-0N) was designed by replacing nitrogen atom of lactam in PL with carbon atom to increase its electrophilicity and thus anti-inflammatory activity. It was found that PL-0N is more potent than the parent compound in suppressing lipopolysaccharide (LPS)-induced secretion of nitric oxide and prostaglandin E2 as well as expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW264.7 macrophages. Mechanistic investigation implies that PL-0N exerts anti-inflammatory activity through inhibition of LPS-induced NF-κB transduction pathway, down-regulation of LPS-induced MAPKs activation and impairment of proteasomal activity, but also enhancement of LPS-induced autophagy; the inhibition of NF-κB by PL-0N is achieved at various stages by: (i) preventing phosphorylation of IKKα/ß, (ii) stabilizing the suppressor protein IκBα, (iii) interfering with the nuclear translocation of NF-κB, and (iv) inhibiting the DNA-binding of NF-κB. These data indicate that nitrogen-atom-lacking pattern is a successful strategy to improve anti-inflammatory property of PL, and that the novel molecule, PL-0N may be served as a promising lead for developing natural product-directed anti-inflammatory agents.

An effective strategy to develop active cinnamic acid-directed antioxidants based on elongating the conjugated chains.

To optimize antioxidant activity and lipophilicity of cinnamic acid derivatives (CAs) including ferulic acid, sinapic acid, 3,4-dimethoxycinnamic acid, and p-hydroxycinnamic acid, four analogs bearing an additional double bond between their aromatic ring and propenoic acid moiety were designed and synthesized based on the conjugated chain elongation strategy. The antioxidant performance of the CAs were investigated by 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-scavenging, ferric reducing/antioxidant power, cyclic voltammetry, DNA strand breakage-inhibiting and anti-haemolysis activity assays. It was found that CAs with elongation of conjugated chains display increased DPPH-scavenging, DNA strand breakage-inhibiting and anti-haemolysis activities as compared to their parent molecules, due to their improved hydrogen atom-donating ability and lipophilicity. Overall, this work highlights an effective strategy to develop potential CA-directed antioxidants by elongating their conjugated chain.