Integration of caveolin-mediated cytosolic delivery and enzyme-responsive releasing of squalenoyl nanoparticles enhance the anti-cancer efficacy of chidamide in pancreatic cancer.

We explored the potential of overcoming the dense interstitial barrier in pancreatic cancer treatment by enhancing the uptake of hydrophilic chemotherapeutic drugs. In this study, we synthesized the squalenoyl-chidamide prodrug (SQ-CHI), linking lipophilic squalene (SQ) with the hydrophilic antitumor drug chidamide (CHI) through a trypsin-responsive bond. Self-assembled nanoparticles with sigma receptor-bound aminoethyl anisamide (AEAA) modification, forming AEAA-PEG-SQ-CHI NPs (A-C NPs, size 116.6 ± 0.4 nm), and reference nanoparticles without AEAA modification, forming mPEG-SQ-CHI NPs (M-C NPs, size 88.3 ± 0.3 nm), were prepared. A-C NPs exhibited significantly higher in vitro CHI release (74.7 %) in 0.5 % trypsin medium compared to release (20.2 %) in medium without trypsin. In vitro cell uptake assays revealed 3.6 and 2.3times higher permeation of A-C NPs into tumorspheres of PSN-1/HPSC or CFPAC-1/HPSC, respectively, compared to M-C NPs. Following intraperitoneal administration to subcutaneous tumor-bearing nude mice, the A-C NPs group demonstrated significant anti-pancreatic cancer efficacy, inducing cancer cell apoptosis and inhibiting proliferation in vivo. Mechanistic studies revealed that AEAA surface modification on nanoparticles promoted intracellular uptake through caveolin-mediated endocytosis. This nanoparticle system presents a novel therapeutic approach for pancreatic cancer treatment, offering a delivery strategy to enhance efficacy through improved tumor permeation, trypsin-responsive drug release, and specific cell surface receptor-mediated intracellular uptake.

Albumin conjugation promotes arsenic trioxide transport through alkaline phosphatase-associated transcytosis in MUC4 wildtype pancreatic cancer cells.

Pancreatic cancer (PC) has a poor prognosis due to chemotherapy resistance and unfavorable drug transportation. Albumin conjugates are commonly used as drug carriers to overcome these obstacles. However, membrane-bound glycoprotein mucin 4 (MUC4) has emerged as a promising biomarker among the genetic mutations affecting albumin conjugates therapeutic window. Human serum albumin-conjugated arsenic trioxide (HSA-ATO) has shown potential in treating solid tumors but is limited in PC therapy due to unclear targets and mechanisms. This study investigated the transport mechanisms and therapeutic efficacy of HSA-ATO in PC cells with different MUC4 mutation statuses. Results revealed improved penetration of ATO into PC tumors through conjugated with HSA. However, MUC4 mutation significantly affected treatment sensitivity and HSA-ATO uptake both in vitro and in vivo. Mutant MUC4 cells exhibited over ten times higher IC50 for HSA-ATO and approximately half the uptake compared to wildtype cells. Further research demonstrated that ALPL activation by HSA-ATO enhanced transcytosis in wildtype MUC4 PC cells but not in mutant MUC4 cells, leading to impaired uptake and weaker antitumor effects. Reprogramming the transport process holds potential for enhancing albumin conjugate efficacy in PC patients with different MUC4 mutation statuses, paving the way for stratified treatment using these delivery vehicles.

The suppressive efficacy of THZ1 depends on KRAS mutation subtype and is associated with super-enhancer activity and the PI3K/AKT/mTOR signalling in pancreatic ductal adenocarcinoma: A hypothesis-generating study.

BACKGROUND: Inhibition of CDK7, a potent transcription regulator, may bring new hope for treating pancreatic ductal adenocarcinoma (PDAC), which is featured by large genetic heterogeneity and abundant KRAS mutations. This investigation aimed at exploring the discrepant efficacies of THZ1, a small-molecule covalent CDK7 inhibitor, on PDACs with different KRAS mutations and the underlying mechanisms. METHODS: Associations of CDK7 expression with survival by KRAS mutations were first assessed. Effects of THZ1 on PDAC by different KRAS mutations were then investigated in vitro and in vivo. Moreover, the effects of THZ1 on gene transcription and phosphorylation of RNA polymerase II (RNAPOLII) in different KRAS mutant PDACs were assessed, and the effect of THZ1 on super-enhancer activity was evaluated using chromatin immunoprecipitation sequencing. Lastly, the effects of THZ1 on the binding of H3K27ac to PIK3CA and on the PI3K/AKT/mTOR signalling were analysed. RESULTS: High CDK7 expression was significantly linked to worse survival within PDAC patients carrying KRAS-G12V mutation but not in those with KRAS-G12D mutation. The apoptosis-inducing effect of THZ1 was markedly stronger in KRAS-G12V PDAC than KRAS-G12D cancer. THZ1 significantly inhibited the growth of xenograft tumour with KRAS-G12V mutation, and the inhibition was markedly stronger than for KRAS-G12D tumour. In mini-cell-derived xenograft (CDX) models, THZ1 significantly suppressed KRAS-G12V PDAC but not KRAS-G12D cancer. THZ1 significantly suppressed the phosphorylation of RNAPOLII, and this effect was stronger in KRAS-G12V PDAC (especially at ser5). KRAS-G12V PDAC had more H3K27ac-binding super-enhancers, and the inhibition of THZ1 on super-enhancer activity was also stronger in KRAS-G12V PDAC. Furthermore, THZ1 significantly weakened the binding of H3K27ac to PIK3CA in KRAS-G12V PDAC. THZ1 significantly suppressed the PI3K/AKT/mTOR pathway and its downstream markers, and this effect was stronger in KRAS-G12V cells. CONCLUSIONS: In this hypothesis-generating study, THZ1 might selectively inhibit certain PDACs with KRAS-G12V mutation more potently compared with some other PDACs with KRAS-G12D mutation, which might be associated with its effect on super-enhancer activity and the PI3K/AKT/mTOR signalling. Our findings might offer novel key clues for the precise management of PDAC and important evidence for future targeted trial design. HIGHLIGHTS: THZ1 had a stronger effect on PDAC-bearing KRAS-G12V mutation than G12D mutation. Suppressive effect of THZ1 on phosphorylation of RNAPOLII was stronger in KRAS-G12V than KRAS-G12D PDAC. Inhibition of THZ1 on super-enhancer activity and H3K27ac binding to PIK3CA was stronger in KRAS-G12V PDAC. Suppressive effect of THZ1 on PI3K/AKT/mTOR pathway was stronger in KRAS-G12V PDAC.

Preliminary delivery efficiency prediction of nanotherapeutics into crucial cell populations in bone marrow niche.

Several crucial stromal cell populations regulate hematopoiesis and malignant diseases in bone marrow niches. Precise regulation of these cell types can remodel niches and develop new therapeutics. Multiple nanocarriers have been developed to transport drugs into the bone marrow selectively. However, the delivery efficiency of these nanotherapeutics into crucial niche cells is still unknown, and there is no method available for predicting delivery efficiency in these cell types. Here, we constructed a three-dimensional bone marrow niche composed of three crucial cell populations: endothelial cells (ECs), mesenchymal stromal cells (MSCs), and osteoblasts (OBs). Mimetic niches were used to detect the cellular uptake of three typical drug nanocarriers into ECs/MSCs/OBs in vitro. Less than 5% of nanocarriers were taken up by three stromal cell types, and most of them were located in the extracellular matrix. Delivery efficiency in sinusoidal ECs, arteriole ECs, MSCs, and OBs in vivo was analyzed. The correlation analysis showed that the cellular uptake of three nanocarriers in crucial cell types in vitro is positively linear correlated with its delivery efficiency in vivo. The delivery efficiency into MSCs was remarkably higher than that into ECs and OBs, no matter what kind of nanocarrier. The overall efficiency into sinusoidal ECs was greatly lower than that into arteriole ECs. All nanocarriers were hard to be delivered into OBs (<1%). Our findings revealed that cell tropisms of nanocarriers with different compositions and ligand attachments in vivo could be predicted via detecting their cellular uptake in bone marrow niches in vitro. This study provided the methodology for niche-directed nanotherapeutics development.

Acute toxicities of intravenous, intraperitoneal, or intratumoral injection of natural killer cells in human pancreatic adenocarcinoma-bearing mice: Randomized study.

AIMS: To investigate the possible acute toxicities and pathological changes associated with intravenous, intraperitoneal, or intratumoral injection of natural killer (NK) cells in mice subcutaneously bearing human pancreatic adenocarcinoma (PaC). METHODS: 100 NPG tumor-bearing mice (50/sex) were engrafted subcutaneously with human PaC BXPC-3 cells 9 days before administration. They were randomly divided into 10 groups with 5 males and 5 females in each group. Mice in Group 1 were given sodium chloride intravenously as vehicle control, and mice in Groups 2-4 human peripheral blood-derived NK cells intravenously at doses of 2 × 107, 1 × 108, and 5 × 108 cells/kg, respectively; mice in Groups 5-7 were injected with NK cells intraperitoneally at doses of 2 × 107, 1 × 108, and 5 × 108 cells/kg, respectively, and mice in Groups 8-10 with NK cells intratumorally at doses of 4 × 103, 2 × 104, and 1 × 105 cells/mm3, respectively. Each group was given a single dose; the mice were observed clinically, and body weight, food intake, blood biochemistry, and tumor volume were measured. On Day 15, the mice were euthanized for gross anatomy and histopathology. RESULTS: On planned euthanasia, in Groups 2-4 no gross or microscopic pathological changes related to cells injection were found; in Groups 5-7 mice of both sexes showed a decrease in extramedullary hematopoiesis of spleen, and at the dose of 5 × 108 cells/kg, mice of both sexes showed an increase in the composition of spleen white pulp cells. In Groups 8-10, mice of both sexes at doses of 4 × 103 and 1 × 105 cells/mm3 and female mice at the dose of 2 × 104 cells/mm3 showed a decrease in extramedullary hematopoiesis of spleen, and female mice at a dose of 4 × 103 cells/mm3 and mice of both sexes at doses of ≥ 2 × 104 cells/mm3 showed an increase in the composition of spleen white pulp cells; perivascular/peribronchiolar inflammatory cell infiltration in lung and bronchus was observed in mice of both sexes at doses of ≥ 2 × 104 cells/mm3, and inflammatory cell infiltration in liver was observed in mice of both sexes at a dose of 1 × 105 cells/mm3. No other abnormal changes with toxicological significance in clinical observation, body weight, food intake, or blood biochemistry were observed in each group. CONCLUSIONS: In our study intravenous injection appears the safest way to give NK cells to human PaC-bearing mice. Using intraperitoneal or intratumoral administration, spleen, liver, and lung were the most often affected organs, albeit with mostly mild pathological changes.

A Three-Dimensional Technique for The Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells.

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Relaxin-encapsulated polymeric metformin nanoparticles remodel tumor immune microenvironment by reducing CAFs for efficient triple-negative breast cancer immunotherapy.

Cancer-associated fibroblasts (CAFs) are one of the most abundant stromal cells in the tumor microenvironment which mediate desmoplastic response and are the primary driver for an immunosuppressive microenvironment, leading to the failure of triple-negative breast cancer (TNBC) immunotherapy. Therefore, depleting CAFs may enhance the effect of immunotherapy (such as PD-L1 antibody). Relaxin (RLN) has been demonstrated to significantly improve transforming growth factor-ß (TGF-ß) induced CAFs activation and tumor immunosuppressive microenvironment. However, the short half-life and systemic vasodilation of RLN limit its in vivo efficacy. Here, plasmid encoding relaxin (pRLN) to locally express RLN was delivered with a new positively charged polymer named polymeric metformin (PolyMet), which could increase gene transfer efficiency significantly and have low toxicity that have been certified by our lab before. In order to improve the stability of pRLN in vivo, this complex was further formed lipid poly-γ-glutamic acid (PGA)/PolyMet-pRLN nanoparticle (LPPR). The particle size of LPPR was 205.5 ± 2.9 nm, and the zeta potential was +55.4 ± 1.6 mV. LPPR displayed excellent tumor penetrating efficacy and weaken proliferation of CAFs in 4T1luc/CAFs tumor spheres in vitro. In vivo, it could reverse aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and remove the physical barrier to reshape the tumor stromal microenvironment, which enabled a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in immunosuppressive cells infiltration. Thus, LPPR was observed retarded tumor growth by itself in the 4T1 tumor bearing-mouse, and the reshaped immune microenvironment further led to facilitate antitumor effect when it combined with PD-L1 antibody (aPD-L1). Altogether, this study presented a novel therapeutic approach against tumor stroma using LPPR to achieve a combination regimen with immune checkpoint blockade therapy against the desmoplastic TNBC model.

Chemical profiling and identification of anti-osteoporosis chemical-markers of Cinnamomum cassia (L.) presl extracts using GC-MS and spectrum-activity analyses.

Cinnamomum cassia (L.) Presl (cinnamon), an important folk medicine is widely used to prevent osteoporosis for long time in China. Our study aimed to investigate the anti-osteoporosis activity and mechanisms of cinnamon extracts obtained by supercritical CO2 extraction (SFE) and identify activity associated chemical components by gas chromatography-mass spectrometry. The cinnamon SFE exhibited superior anti-osteoporosis efficacy in an ovariectomised mice model to common alcohol extracts. It could induce calcified nodules and ALP activity, upregulate the mRNA expression of ALP, BMP-2, and RUNX2 in MC3T3-E1 cells. The major chemical classes of cinnamon extracts were alcohol esters (28.2%), and terpenes (16.1%). The spectrum-activity analysis indicated that the potential chemical-markers of extracts could be (E)-Cinnamaldehyde, γ-Sitosterol, and (Z, Z)-9,12-Octadecadienoic acid, which could induce the proliferation and ALP activity in MC3T3-E1 cells. Our study revealed the promising applications of the cinnamon SFE in prevention of osteoporosis, and identified its anti-osteoporosis associated compounds.

N-Butanol Extract of Modified You-Gui-Yin Attenuates Osteoclastogenesis and Ameliorates Osteoporosis by Inhibiting RANKL-Mediated NF-κB Signaling.

Postmenopausal Osteoporosis (PMOP) is the most prevalent primary osteoporosis, attributable to an imbalance in osteoblast and osteoclast activity. Modified You-Gui-Yin (MYGY), a traditional Chinese herbal formula, is able to effectively treat PMOP, while the critical components and pharmacological mechanisms of MYGY are still unclear. In this study, we aimed to investigate the therapeutic effects and underlying mechanisms of N-butanol extract of MYGY (MYGY-Nb) in ovariectomized (OVX)-induced osteoporosis mice. Histological staining and micro-computed tomography (µCT) analysis showed that MYGY-Nb was more effective in the suppression of OVX-induced bone loss than MYGY original formula. Subsequently, liquid chromatography and mass spectrometry analysis identified 16 critical compounds of MYGY-Nb and some of them are reported to affect osteoclast functions. Furthermore, in vivo and in vitro experiments demonstrated that MYGY-Nb significantly attenuated osteoclastogenesis by down-regulating RANKL-mediated NF-κB signaling. In conclusion, our study indicated that MYGY-Nb suppresses NF-κB signaling and osteoclast formation to mitigate bone loss in PMOP, implying that MYGY-Nb and its compounds are potential candidates for development of anti-PMOP drugs.

Reversing multi-drug resistance by polymeric metformin to enhance antitumor efficacy of chemotherapy.

Multi-drug resistance (MDR) in breast cancer poses a great threat to chemotherapy. The expression and function of the ATP binding cassette (ABC) transporter are the major cause of MDR. Herein, a linear polyethylene glycol (PEI) conjugated with dicyandiamide, which called polymeric metformin (PolyMet), was successfully synthesized as a simple and biocompatible polymer of metformin. PolyMet showed the potential to reverse MDR by inhibiting the efflux of the substrate of ATP-binding cassette (ABC) transporter from DOX resistant MCF-7 cells (MCF-7/DOX). To test its MDR reversing effect, PolyMet was combined with DOX to treat mice carrying MCF-7/DOX xenografts. In order to decrease the toxicities of DOX and delivery PolyMet and DOX to tumor at the same time, PolyMet was complexed with poly-γ-glutamic acid-doxorubicin (PGA-DOX) electrostatically at the optimal ratio of 2:3, which were further coated with lipid membrane to form lipid/PolyMet-(PGA-DOX) nanoparticles (LPPD). The particle size of LPPD was 165.8 nm, and the zeta potential was +36.5 mV. LPPD exhibited favorable cytotoxicity and cellular uptake in MCF-7/DOX. Meanwhile, the bioluminescence imaging and immunohistochemical analysis indicated that LPPD effectively conquered DOX-associated MDR by blocking ABC transporters (ABCB1 and ABCC1) via PolyMet. Remarkably, LPPD significantly inhibited the tumor growth and lowered the systemic toxicity in a murine MCF-7/DOX tumor model. This is the first time to reveal that PolyMet can enhance the anti-tumor efficacy of DOX by dampening ABC transporters and activating the AMPK/mTOR pathway, which is a promising strategy for drug-resistant breast cancer therapy.

The osteogenic niche-targeted arsenic nanoparticles prevent colonization of disseminated breast tumor cells in the bone.

Up to 70% of patients with late-stage breast cancer have bone metastasis. Current treatment regimens for breast cancer bone metastasis are palliative with no therapeutic cure. Disseminated tumor cells (DTCs) colonize inside the osteogenic niches in the early stage of bone metastasis. Drug delivery into osteogenic niches to inhibit DTC colonization can prevent bone metastasis from entering its late stage and therefore cure bone metastasis. Here, we constructed a 50% DSS6 peptide conjugated nanoparticle to target the osteogenic niche. The osteogenic niche was always located at the endosteum with immature hydroxyapatite. Arsenic-manganese nanocrystals (around 14 nm) were loaded in osteogenic niche-targeted PEG-PLGA nanoparticles with an acidic environment-triggered arsenic release. Arsenic formulations greatly reduced 4T1 cell adhesion to mesenchymal stem cells (MSCs)/preosteoblasts (pre-OBs) and osteogenic differentiation of osteoblastic cells. Arsenic formulations also prevented tumor cell colonization and dormancy via altering the direct interaction between 4T1 cells and MSCs/pre-OBs. The chemotactic migration of 4T1 cells toward osteogenic cells was blocked by arsenic in mimic 3D osteogenic niche. Systemic administration of osteogenic niche-targeted arsenic nanoparticles significantly extended the survival of mice with 4T1 syngeneic bone metastasis. Our findings provide an effective approach for osteogenic niche-specific drug delivery and suggest that bone metastasis can be effectively inhibited by blockage of tumor cell colonization in the bone microenvironment.

Fractions of Shen-Sui-Tong-Zhi Formula Enhance Osteogenesis Via Activation of ß-Catenin Signaling in Growth Plate Chondrocytes.

Background: Shen-sui-tong-zhi formula (SSTZF) has been used to treat osteoporosis for decades and shows excellent clinical efficacy. This article aims to explore the optimal anti-osteoporotic ingredient and its precise mechanisms in mice models. Methods: In this study, we first screened the optimal anti-osteoporosis fraction of SSTZF extract in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old female C57BL/6J mice were administrated with each fraction of SSTZF. At 10 weeks after ovariectomy (OVX), femurs were collected for tissue analyses, including histology, micro-CT, biomechanical tests, and immunohistochemistry for ALP, FABP4, and ß-catenin. Additionally, we also evaluated the mRNA expression level of ALP and FABP4 and the protein expression level of ß-catenin after being treated with SSTZF extract in C3H10T1/2 cells. Moreover, we investigated the anti-osteoporosis effect of SSTZF extract on mice with ß-catenin conditional knockout in growth plate chondrocytes (ß-catenin Gli1ER mice) through µCT, histology, and immunohistochemistry analyzes. Results: At 10 weeks after treatment, osteoporosis-like phenotype were significantly ameliorated in SSTZF n-butanol extract (SSTZF-NB) group mice, as indicated by increased trabecular bone area and ALP content, and decreased lipid droplet area and FABP4 content. No such improvements were observed after being treated with other extracts, demonstrating that SSTZF-NB is the optimal anti-osteoporosis fraction. Additionally, the elevated ß-catenin was revealed in both OVX mice and C3H10T1/2 cells with SSTZF-NB administered. Furthermore, a significant osteoporosis-like phenotype was observed in ß-catenin Gli1ER mice as expected. However, SSTZF-NB failed to rescue the deterioration in ß-catenin Gli1ER mice, no significant re-upregulated ALP and downregulated FABP4 were observed after being treated with SSTZF-NB, demonstrating that SSTZF-NB prevents bone loss mainly via ß-catenin signaling. Conclusion: SSTZF-NB enhances osteogenesis mainly via activation of ß-catenin signaling in growth plate chondrocytes. SSTZF-NB is the optimal anti-osteoporosis fraction of SSTZF and it can be considered a salutary alternative therapeutic option for osteoporosis.

Valtrate as a novel therapeutic agent exhibits potent anti-pancreatic cancer activity by inhibiting Stat3 signaling.

BACKGROUND: Valtrate is a novel epoxy iridoid ester isolated from Chinese herbal medicine Valeriana jatamansi Jones with anti-proliferative activity against various human cancer cell lines. However, its efficacy and molecular mechanisms against pancreatic cancer (PC) cells are largely unclear. PURPOSE: To investigate the anti-cancer effects of valtrate on PC cell lines and its underlying mechanisms. METHODS: MTT assay was first performed to detect the effect of valtrate on cell viability in human PC cell lines and normal pancreatic epithelial cells HPDE. Cell apoptosis and cycle phase assay were detected by flow cytometry. The relative mRNA expressions of Bax, Bcl-2, c-Myc, and CyclinB1 were tested by quantitative PCR (qPCR) assay. The expression of relative proteins was detected by Western blotting (WB). A PANC-1luc cells xenograft mouse model in nu/nu female mice was used to elucidate the effect of valtrate on tumor growth in vivo. RESULTS: Valtrate significantly inhibited the growth of PC cells without affecting the growth of normal pancreatic epithelial cells HPDE, induced significant apoptosis and cell cycle arrest in G2/M phase. Moreover, valtrate inhibited the tumor growth of PC cell PANC-1 in xenograft mice by 61%. Further mechanism study demonstrated that valtrate could increase the expression level of Bax, suppress Bcl-2 as well as c-Myc and Cyclin B1, inhibit the transcriptional activity of Stat3, while valtrate decreased the expression level of Stat3 and phosphated-Stat3 (Tyr705) and induced the high molecular aggregation of Stat3. Molecular docking analysis predicted that valtrate might interact with Cys712 of Stat3 protein. Valtrate could also induce a transient depleted intracellular glutathione (GSH) level and increased reactive oxygen species (ROS). NAC (N-acetylcysteine), a reducer reversed valtrate-induced the depletion of Stat3, p-Stat3, c-Myc, and Cyclin B1. CONCLUSION: Valtrate exerts anti-cancer activity against PC cells by directly targeting Stat3 through a covalent linkage to inhibit Stat3 activity, which causes apoptosis and cell cycle arrest.

Wing rapid responses and aerodynamics of fruit flies during headwind gust perturbations.

Insects are the main source of inspiration for flapping-wing micro air vehicles (FWMAVs). They frequently encounter wind gust perturbations in natural environments, and effectively cope with these perturbations. Here, we investigated the rapid gust response of flies to instruct the gust stability design of FWMAVs. A novel method to produce impulsive wind gusts that lasted less than 30 ms was designed to observe flies' rapid responses. Headwind gust perturbations were imposed on 14 tethered fruit flies, and the corresponding wing motions during perturbations were recorded by three high-speed cameras. The numerical simulation method was then applied to analyze aerodynamic forces and moments induced by the changes in wing kinematics. Results shows that flies mainly utilize three strategies against headwind gust perturbations, including decreasing the magnitude of stroke positional angle at ventral stroke reversal, delayed rotation and making the deviation angles in upstroke and downstroke closer (i.e. the wing tip trajectories of upstroke and downstroke tend be closer). Consequently, flies resist increments in lift and drag induced by the headwind gusts. However, flies seem to care little about changes in pitch moment in tethered conditions. These results provide useful suggestions for the stability control of FWMAVs during headwind gust perturbations.

Metabolic Profiling of Formalin-Fixed Paraffin-Embedded Tissues Discriminates Normal Colon from Colorectal Cancer.

Accumulating evidence suggests that metabolic reprogramming has a critical role in carcinogenesis and tumor progression. The usefulness of formalin-fixed paraffin-embedded (FFPE) tissue material for metabolomics analysis as compared with fresh frozen tissue material remains unclear. LC/MS-MS-based metabolomics analysis was performed on 11 pairs of matched tumor and normal tissues in both FFPE and fresh frozen tissue materials from patients with colorectal carcinoma. Permutation t test was applied to identify metabolites with differential abundance between tumor and normal tissues. A total of 200 metabolites were detected in the FFPE samples and 536 in the fresh frozen samples. The preservation of metabolites in FFPE samples was diverse according to classes and chemical characteristics, ranging from 78% (energy) to 0% (peptides). Compared with the normal tissues, 34 (17%) and 174 (32%) metabolites were either accumulated or depleted in the tumor tissues derived from FFPE and fresh frozen samples, respectively. Among them, 15 metabolites were common in both FFPE and fresh frozen samples. Notably, branched chain amino acids were highly accumulated in tumor tissues. Using KEGG pathway analyses, glyoxylate and dicarboxylate metabolism, arginine and proline, glycerophospholipid, and glycine, serine, and threonine metabolism pathways distinguishing tumor from normal tissues were found in both FFPE and fresh frozen samples. This study demonstrates that informative data of metabolic profiles can be retrieved from FFPE tissue materials. IMPLICATIONS: Our findings suggest potential value of metabolic profiling using FFPE tumor tissues and may help to shape future translational studies through developing treatment strategies targeting metabolites.

Night-Shift Work Duration and Risk of Colorectal Cancer According to IRS1 and IRS2 Expression.

BACKGROUND: We hypothesized that the risk of colorectal cancer in night-shift workers might be different according to insulin receptor substrate status. METHODS: Among 77,470 eligible women having night work assessed in the Nurses' Health Study, we documented a total of 1,397 colorectal cancer cases, of which 304 or 308 had available data on IRS1 and IRS2, respectively. We used duplication-method Cox proportional hazards regression analysis for competing risks to calculate HRs and 95% confidence intervals (CI) for each colorectal cancer subtype. We measured tumor IRS1 or IRS2 expression by immunohistochemistry (IHC). RESULTS: Compared with women who never worked night shifts, those working ≥15 years night shifts had a marginal trend of increased overall risk of colorectal cancer (P trend = 0.06; multivariable HR = 1.20; 95% CI, 0.99-1.45). Longer duration of night-shift work was associated with a higher risk of IRS2-positive tumors (multivariable HR = 2.69; 95% CI, 1.48-4.89; P trend = 0.001, ≥15 years night shifts vs. never) but not with IRS2-negative tumors (multivariable HR = 0.90; 95% CI, 0.54-1.51; P trend = 0.72; P heterogeneity for IRS2 = 0.008). Similarly, the corresponding multivariable HRs were 1.81 for IRS1-positive tumors (95% CI, 0.94-3.48; P trend = 0.06) and 1.13 for IRS1-negative tumors (95% CI, 0.71-1.80; P trend = 0.56; P heterogeneity for IRS1 = 0.02). CONCLUSIONS: Our molecular pathologic epidemiology data suggest a potential role of IRS in mediating carcinogenesis induced by night-shift work. IMPACT: Although these findings need validation, rotating night shift might increase colorectal cancer risk in women with abnormal insulin receptor pathways.

Coix Seed Extract Enhances the Anti-Pancreatic Cancer Efficacy of Gemcitabine through Regulating ABCB1- and ABCG2-Mediated Drug Efflux: A Bioluminescent Pharmacokinetic and Pharmacodynamic Study.

A deep insight into the function and kinetics of ATP-binding cassette (ABC) transporters may aid in the development of pharmaceutics that can minimize the particular facet of chemo-resistance. We utilized bioluminescence imaging to monitor the ABC transporter mediated intracellular drug efflux function. We also investigated the potential association between the intracellular bioluminescent pharmacokinetic profiles and the anti-tumor efficacy of the coix seed extract and gemcitabine against pancreatic cancer cells in vitro and in vivo. The bioluminescent pharmacokinetic parameters and pharmacodynamic index (IC50 and TGI) were determined. The expression levels ABCB1 and ABCG2 were assessed. Results showed that coix seed extract could synergistically enhance the anti-cancer efficacy of gemcitabine (p < 0.05). Meanwhile coix seed extract alone or in combination with gemcitabine could significantly increase the AUCluc while decreasing the Kluc (p < 0.01). Western blot and immunohistochemistry assay demonstrated that coix seed extract could significantly mitigate gemcitabine-induced upregulation of ABCB1 and ABCG2 protein. The Pearson correlation analysis demonstrated that the bioluminescent pharmacokinetic parameters and pharmacodynamic index have strong association in vitro and in vivo. In conclusion coix seed extract could augment the efficacy of gemcitabine therapy in pancreatic cancer cells may at least partly due to the alteration of ABC transporter-mediated drug efflux function.

UPLC­MS/MS­based metabolomic characterization and comparison of pancreatic adenocarcinoma tissues using formalin­fixed, paraffin­embedded and optimal cutting temperature­embedded materials.

The purpose of the present study was to compare metabolites from formalin­fixed and paraffin­embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)­embedded pancreatic tissue blocks. Thus, ultra­performance liquid chromatograph­mass spectrometry/mass spectrometry­based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT­embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least­squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N­acetylaspartate and creatinine, was significantly different in both OCT­embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super­categories to which they belonged exhibited no significant differences between FFPE and OCT­embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT­embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.

In Vitro and In Situ Characterization of the Intestinal Absorption of Capilliposide B and Capilliposide C from Lysimachia capillipes Hemsl.

The goal of this investigation was to determine the processes and mechanism of intestinal absorption for capilliposide B (CAPB) and capilliposide C (CAPC) from the Chinese herb, Lysimachia capillipes Hemsl. An analysis of basic parameters, such as drug concentrations, time, and behavior in different intestinal segments was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS). The susceptibility of CAPB and CAPC to various inhibitors such as P-glycoprotein (P-gp) inhibitor (verapamil); multidrug resistance-associated protein 2 (MRP2) inhibitor (indomethacin); cytochrome P450 protein 3A4 (CYP3A4) inhibitor (ketoconazole); and the co-inhibitor of P-gp, MRP2 and CYP3A4 (cyclosporine A) were assessed using both caco-2 cell monolayer and single-pass intestinal perfusion (SPIP) models. As a result, CAPB and CAPC are both poorly absorbed in the intestines and exhibited segment-dependent permeability. The intestinal permeability of CAPB and CAPC were significantly increased by the co-treatment of verapamil, indomethacin. In addition, the intestinal permeability of CAPB was also enhanced by ketoconazole and cyclosporine A. It can be concluded that the intestinal absorption mechanisms of CAPB and CAPC involve processes such as facilitated passive diffusion, efflux transporters, and enzyme-mediated metabolism. Both CAPB and CAPC are suggested to be substrates of P-gp and MRP2. However, CAPB may interact with the CYP3A4 system.

Intranasal administration of pullulan-based nanoparticles for enhanced delivery of adriamycin into the brain: in vitro and in vivo evaluation.

Intranasal (i.n.) administration is an efficient route for enhancing drug delivery to the brain, bypassing the blood-brain barrier (BBB) and eliminating systemic side effects. The purpose of this study was to investigate the nose-to-brain delivery efficiency of adriamycin (ADM) loaded in cholesterol-modified pullulan self-assembled nanoparticles (CHSP-SAN) via i.n. administration. The prepared nanodrugs (ADM-CHSP-SAN) were characterized as uniform size (112.8±1.02 nm), high drug loading capacity (7.65±0.58 %), and sustained release. CHSP-SAN showed good biocompatibility and low toxicity on HBMEC and C6 cells. The enhanced delivery of ADM across the BBB with CHSP-SAN was demonstrated by the reduced half maximal inhibitory concentration (IC50) value and the increased apoptosis proportion of C6 cells. The pharmacokinetics of ADM-CHSP-SAN was accessed by cerebral microdialysis technique. The pharmacokinetic results showed higher peak concentration (Cmax), area under the curve (AUC0-12h) and shorter peak time (Tmax) after i.n. administration that after intravenous (i.v.) administration. The i.n. administration of CHSP-SAN greatly increased ADM availability in cerebral tissue compared to that of ADM solution. Collectively, CHSP-SAN strikingly increased ADM transport across the BBB and improved its availability in brain via i.n. administration.