4-Acetylantroquinonol B induced DNA damage response signaling and apoptosis via suppressing CDK2/CDK4 expression in triple negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) has a more aggressive phenotype and poorer prognosis than hormone receptor (HR+) and human epidermal growth factor receptor (HER2 -) subtypes. Inhibition of cyclin-dependent kinase (CDK)4 and CDK6 was successful in patients with advanced metastatic HR+/HER2- breast cancer, but those with TNBC exhibited low or no response to this therapeutic approach. This study investigated the dual therapeutic targeting of CDK2 and CDK4 by using 4-acetyl-antroquinonol B (4-AAQB) against TNBC cells. METHODS: We examined the effects of CDK2, CDK4, and CDK6 inhibition through 4-AAQB treatment on TNBC cell lines and established an orthotropic xenograft mouse model to confirm the in vitro results of inhibiting CDK2, CDK4, and CDK6 by 4-AAQB treatment. RESULTS: High expression and alteration of CDK2 and CDK4 but not CDK6 significantly correlated with poor overall survival of patients with breast cancer. CDK2 and CDK4 were positively correlated with damage in DNA replication and repair pathways. Docking results indicated that 4-AAQB was bound to CDK2 and CDK4 with high affinity. Treatment of TNBC cells with 4-AAQB suppressed the expression of CDK2 and CDK4 in vitro. Additionally, 4-AAQB induced cell cycle arrest, DNA damage, and apoptosis in TNBC cells. In vivo study results confirmed that the anticancer activity of 4-AAQB suppressed tumor growth through the inhibition of CDK2 and CDK4. CONCLUSION: The expression level of CDK2 and CDK4 and DNA damage response (DDR) signaling are prominent in TNBC cell cycle regulation. Thus, 4-AAQB is a potential agent for targeting CDK2/4 and DDR in TNBC cells.

Pharmacological bypass of NAD+ salvage pathway protects neurons from chemotherapy-induced degeneration.

Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD+) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD+, nicotinamide mononucleotide (NMN), rather than loss of NAD+, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD+ generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD+ is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.

In Situ Magnetic Control of Macroscale Nanoligand Density Regulates the Adhesion and Differentiation of Stem Cells.

Developing materials with remote controllability of macroscale ligand presentation can mimic extracellular matrix (ECM) remodeling to regulate cellular adhesion in vivo. Herein, we designed charged mobile nanoligands with superparamagnetic nanomaterials amine-functionalized and conjugated with polyethylene glycol linker and negatively charged RGD ligand. We coupled negatively a charged nanoligand to a positively charged substrate by optimizing electrostatic interactions to allow reversible planar movement. We demonstrate the imaging of both macroscale and in situ nanoscale nanoligand movement by magnetically attracting charged nanoligand to manipulate macroscale ligand density. We show that in situ magnetic control of attracting charged nanoligand facilitates stem cell adhesion, both in vitro and in vivo, with reversible control. Furthermore, we unravel that in situ magnetic attraction of charged nanoligand stimulates mechanosensing-mediated differentiation of stem cells. This remote controllability of ECM-mimicking reversible ligand variations is promising for regulating diverse reparative cellular processes in vivo.

The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Early steps of gene expression are a composite of promoter recognition, promoter activation, RNA synthesis and RNA processing, and it is known that SUMOylation, a post-translational modification, is involved in transcription regulation. We previously found that SUMO-1 marks chromatin at the proximal promoter regions of some of the most active housekeeping genes during interphase in human cells, but the SUMOylated targets on the chromatin remained unclear. In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing. Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected. This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.

Promoters active in interphase are bookmarked during mitosis by ubiquitination.

We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis.

Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes.

SUMOylation of transcription factors and chromatin proteins is in many cases a negative mark that recruits factors that repress gene expression. In this study, we determined the occupancy of Small Ubiquitin-like MOdifier (SUMO)-1 on chromatin in HeLa cells by use of chromatin affinity purification coupled with next-generation sequencing. We found SUMO-1 localization on chromatin was dynamic throughout the cell cycle. Surprisingly, we observed that from G1 through late S phase, but not during mitosis, SUMO-1 marks the chromatin just upstream of the transcription start site on many of the most active housekeeping genes, including genes encoding translation factors and ribosomal subunit proteins. Moreover, we found that SUMO-1 distribution on promoters was correlated with H3K4me3, another general chromatin activation mark. Depletion of SUMO-1 resulted in downregulation of the genes that were marked by SUMO-1 at their promoters during interphase, supporting the concept that the marking of promoters by SUMO-1 is associated with transcriptional activation of genes involved in ribosome biosynthesis and in the protein translation process.

E339...R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors.

The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism--disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozin-resistant strains. This work provides a new approach to design antiinfluenza drugs.

[Multi-factor Impact Analysis of Grassland Phenology Changes on the Qinghai-Xizang Plateau Based on Interpretable Machine Learning].

The vegetation phenology of the Qinghai-Xizang Plateau is changing significantly in the context of climate change. However, there are many hydrothermal factors affecting the phenology, and few studies have focused on the effects of multiple factors on the phenology of the Qinghai-Xizang Plateau, resulting in a lack of understanding of the mechanisms underlying phenological changes on the Qinghai-Xizang Plateau. In this study, we used remote sensing data interpretation to analyze the spatial and temporal variability of grassland phenology on the Qinghai-Xizang Plateau from 2002 to 2021, focusing on precipitation, temperature, altitude, soil, and other aspects to reveal the dominant factors of phenological variability using an interpretable machine learning method (SHAP) and to quantify the interactive effects of multiple factors on phenology. The results showed that:① The growing season start (SOS) of grasslands on the Qinghai-Xizang Plateau mostly ranged from 110 to 150 d, with 56.32 % of grasslands showing an early SOS trend; the growing season end (EOS) mostly ranged from 290-320 d, with 67.65 % of grasslands showing a delayed EOS trend; and the growing season length (LOS) mostly ranged from 120 to 210 d, with 65.50 % of the grasslands showing a trend towards longer growing season lengths. ② SOS in grasslands on the Qinghai-Xizang Plateau was mainly influenced by moisture conditions, in which soil moisture between 10 and 25 kg·m-2 in the 0-10 cm soil layer in March promoted the advancement of SOS and peaked at approximately 20 kg·m-2. EOS was mainly influenced by temperature, with higher temperatures in September and October having a stronger effect on EOS latency promotion and peaking at over 8 ℃ and -0.5 ℃, respectively. The main influencing factors of LOS were more consistent with SOS, in which soil moisture between 15 and 25 kg·m-2 in the 0-10 cm soil layer in March promoted the prolongation of LOS and peaked at approximately 18 kg·m-2. ③ There was an obvious interactive effect of water and heat and other factors on phenology; after soil moisture reached 20 kg·m-2 in the 0-10 cm soil layer in March, SOS was more advanced in low-precipitation and low-altitude areas. Better moisture conditions were more conducive to EOS delay at temperatures above 0 ℃ in October, and soil moisture in high precipitation areas promoted LOS prolongation more when soil moisture was between 12 and 22 kg·m-2 in 0-10 cm in March. The results also demonstrated that interpretable machine learning methods could provide a new approach to the analysis of the multifactorial effects of phenological change.

Weighted frequent gene co-expression network mining to identify genes involved in genome stability.

Gene co-expression network analysis is an effective method for predicting gene functions and disease biomarkers. However, few studies have systematically identified co-expressed genes involved in the molecular origin and development of various types of tumors. In this study, we used a network mining algorithm to identify tightly connected gene co-expression networks that are frequently present in microarray datasets from 33 types of cancer which were derived from 16 organs/tissues. We compared the results with networks found in multiple normal tissue types and discovered 18 tightly connected frequent networks in cancers, with highly enriched functions on cancer-related activities. Most networks identified also formed physically interacting networks. In contrast, only 6 networks were found in normal tissues, which were highly enriched for housekeeping functions. The largest cancer network contained many genes with genome stability maintenance functions. We tested 13 selected genes from this network for their involvement in genome maintenance using two cell-based assays. Among them, 10 were shown to be involved in either homology-directed DNA repair or centrosome duplication control including the well-known cancer marker MKI67. Our results suggest that the commonly recognized characteristics of cancers are supported by highly coordinated transcriptomic activities. This study also demonstrated that the co-expression network directed approach provides a powerful tool for understanding cancer physiology, predicting new gene functions, as well as providing new target candidates for cancer therapeutics.

Plastispheres as hotspots of microbially-driven methylmercury production in paddy soils.

Microplastics (MPs) as emerging contaminants have accumulated extensively in agricultural ecosystems and are known to exert important effects on biogeochemical processes. However, how MPs in paddy soils influence the conversion of mercury (Hg) to neurotoxic methylmercury (MeHg) remains poorly understood. Here, we evaluated the effects of MPs on Hg methylation and associated microbial communities in microcosms using two typical paddy soils in China (i.e., yellow and red soils). Results showed that the addition of MPs significantly increased MeHg production in both soils, which could be related to higher Hg methylation potential in the plastisphere than in the bulk soil. We found significant divergences in the community composition of Hg methylators between the plastisphere and the bulk soil. In addition, the plastisphere had higher proportions of Geobacterales in the yellow soil and Methanomicrobia in the red soil compared with the bulk soil, respectively; and plastisphere also had more densely connected microbial groups between non-Hg methylators and Hg methylators. These microbiota in the plastisphere are different from those in the bulk soil, which could partially account for their distinct MeHg production ability. Our findings suggest plastisphere as a unique biotope for MeHg production and provide new insights into the environment risks of MP accumulation in agricultural soils.

Analyzing angiogenesis on a chip using deep learning-based image processing.

Angiogenesis, the formation of new blood vessels from existing vessels, has been associated with more than 70 diseases. Although numerous studies have established angiogenesis models, only a few indicators can be used to analyze angiogenic structures. In the present study, we developed an image-processing pipeline based on deep learning to analyze and quantify angiogenesis. We utilized several image-processing algorithms to quantify angiogenesis, including a deep learning-based cell nuclear segmentation algorithm and image skeletonization. This method could quantify and measure changes in blood vessels in response to biochemical gradients using 16 indicators, including length, width, number, and nuclear distribution. Moreover, this procedure is highly efficient for the three-dimensional quantitative analysis of angiogenesis and can be applied to diverse angiogenesis investigations.

Biosensing Amplification by Hybridization Chain Reaction on Phase-Sensitive Surface Plasmon Resonance.

Surface Plasmon Resonance (SPR) is widely used in biological and chemical sensing with fascinating properties. However, the application of SPR to detect trace targets is hampered by non-specific binding and poor signal. A variety of approaches for amplification have been explored to overcome this deficiency including DNA aptamers as versatile target detection tools. Hybridization chain reaction (HCR) is a high-efficiency enzyme-free DNA amplification method operated at room temperature, in which two stable species of DNA hairpins coexist in solution until the introduction of the initiator strand triggers a cascade of hybridization events. At an optimal salt condition, as the concentrations of H1 and H2 increased, the HCR signals were enhanced, leading to signal amplification reaching up to 6.5-fold of the detection measure at 30 min. This feature enables DNA to act as an amplifying transducer for biosensing applications to provide an enzyme-free alternative that can easily detect complex DNA sequences. Improvement of more diverse recognition events can be achieved by integrating HCR with a phase-sensitive SPR (pSPR)-tested aptamer stimulus. This work seeks to establish pSPR aptamer system for highly informative sensing by means of an amplification HCR. Thus, combining pSPR and HCR technologies provide an expandable platform for sensitive biosensing.

Uhrf1 regulates H3K9me2 modification of mTOR to inhibit the effect of autophagy in myocardial ischemia-reperfusion injury.

The regulation of mTOR and the dimethylation of histone H3 on lysine 9 (H3K9me2) H3K9me2 by Uhrf1 and the mechanism of autophagy regulation in myocardial ischemia-reperfusion injury (MIRI) were studied in vivo and in vitro. An in vitro I/R injury model was established using the primary mouse cardiomyocytes treated with H2O2. Subsequent analysis by qRT-PCR, western blot, and immunofluorescence indicated that overexpression of Uhrf1 significantly inhibited apoptosis of the H2O2-treated cardiomyocytes, reduced expression of apoptosis factors caspase-3 and Bax, and increased expression of apoptosis inhibitory factor Bcl-2. Furthermore, Uhrf1 was found to increase cardiomyocyte proliferation and promote the expression of mTOR, while the four expression peaks of H3K9me2 on the mTOR gene were inhibited by overexpression of Uhrf1. The expression of autophagy factors LC3, Beclin-1, and p-mTOR in Uhrf1-overexpressed cardiomyocytes was dramatically increased, and P62 expression was dramatically decreased. When an H3K9me2 inhibitor was added to the Uhrf1-knockdown cardiomyocytes, the expression of mTOR was increased, the expression of LC3, Beclin-1, and p-mTOR was decreased, and P62 expression was significantly increased. In the present study, Uhrf1 exhibits a protective function in MIRI, reducing the apoptosis of cardiomyocytes while increasing their proliferation and viability.

Antrodia salmonea induces apoptosis and enhances cytoprotective autophagy in colon cancer cells.

A traditional Chinese medicinal fungus, Antrodia salmonea (AS), with antioxidant properties is familiar in Taiwan but anti-cancer activity of AS in human colon cancer is ambiguous. Hence, we explored the anti-cancer activity of AS in colon cancer cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that AS showed a remarkable effect on cell viability in colon cancer cells; SW620, HCT116, and HT29. Annexin V/propidium iodide (PI) stained cells indicated that AS induced both early/late apoptosis in SW620 cells. Additionally, cells treated with AS induced caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, mitochondrial dysfunction, and Bcl-2 associated X (Bax)/B-cell lymphoma (Bcl-2) dysregulation. Microtubule- associated protein 1A/1B-light chain 3B (LC3-II) accumulation, sequestosome 1 (p62/SQSTM1) activation, autophagy related 4B cysteine peptidase (ATG4B) inactivation, acidic vesicular organelles (AVOs) formation, and Beclin-1/Bcl-2 dysregulation revealed that AS-induced autophagy. Interestingly, cells pretreated with 3-methyladenine (3-MA) strengthened AS-induced caspase-3/apoptosis. Suppression of apoptosis by z-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not however block AS-induced autophagy, suggesting that autophagy was not attenuated by the AS-induced apoptosis. Application of N-acetylcysteine (NAC) prevented AS-induced cell death, caspase-3 activation, LC3-II accumulation, and AVOs formation, indicating that AS-induced apoptosis and autophagy was mediated by reactive oxygen species (ROS). Furthermore, AS-induced cytoprotective autophagy and apoptosis through extracellular signal-regulated kinase (ERK) signaling cascades. Moreover, in vivo data disclosed that AS inhibited colitis-associated tumorigenesis in azoxymethane (AOM)-dextran sodium sulphate (DSS)-treated mice. For the first time, we report the anti-cancer properties of this potentially advantageous mushroom for the treatment of human colon cancer.

Multi-Layer Reflectivity Calculation Based Meta-Modeling of the Phase Mapping Function for Highly Reproducible Surface Plasmon Resonance Biosensing.

Phase-sensitive surface plasmon resonance biosensors are known for their high sensitivity. One of the technology bottle-necks of such sensors is that the phase sensorgram, when measured at fixed angle set-up, can lead to low reproducibility as the signal conveys multiple data. Leveraging the sensitivity, while securing satisfying reproducibility, is therefore is an underdiscussed key issue. One potential solution is to map the phase sensorgram into refractive index unit by the use of sensor calibration data, via a simple non-linear fit. However, basic fitting functions poorly portray the asymmetric phase curve. On the other hand, multi-layer reflectivity calculation based on the Fresnel coefficient can be employed for a precise mapping function. This numerical approach however lacks the explicit mathematical formulation to be used in an optimization process. To this end, we aim to provide a first methodology for the issue, where mapping functions are constructed from Bayesian optimized multi-layer model of the experimental data. The challenge of using multi-layer model as optimization trial function is addressed by meta-modeling via segmented polynomial approximation. A visualization approach is proposed for assessment of the goodness-of-the-fit on the optimized model. Using metastatic cancer exosome sensing, we demonstrate how the present work paves the way toward better plasmonic sensors.

High-Grade B-Cell Lymphoma (HGBL) with MYC and BCL2 and/or BCL6 Rearrangements Is Predominantly BCL6-Rearranged and BCL6-Expressing in Taiwan.

This study investigated the epidemiological and clinical peculiarities of BCL2 and BCL6 rearrangement in patients with high grade B-cell lymphoma (HGBL) from Taiwan, compared with data from Western countries. Two hundred and eighty-two DLBCL cases from Taipei Medical University-affiliated hospitals (n = 179) and Tri-Service General Hospital (n = 103) were enrolled for this study. From the 282, 47 (16.7%) had MYC translocation; 24 of these harbored concurrent BCL2 and/or BCL6 translocation (double-hit, DH or triple-hit, TH). Twelve DH-HGBL cases had simultaneous MYC and BCL6 translocations, 8 harbored MYC and BCL2 rearrangement, while the remaining 4 patients exhibited TH. Together, 66.7% of DH/TH-HGBL patients were BCL6 rearrangement positive. Among these BCL6-rearranged DH/TH-HGBL patients, only 6 (37.5%) overexpressed MYC and BCL6 proteins simultaneously, indicating that MYC-BCL6 co-overexpression may not be plausible surrogate biomarker for screening BCL6-rearranged DH-HGBL. By the end of year 5, all patients with TH-HGBL, BCL2 DH-HGBL and all but one BCL6 DH-HGBL cases had expired or were lost to follow-up. Progression-free survival (PFS) was longer for the non-DH/TH-HGBL group compared with the DH/TH-HGBL group. While the patients with BCL2 DH-HGBL were lost to follow-up by day 800, their remaining TH-HGBL and BCL6 DH-HGBL peers exhibited very poor PFS, regardless of age strata. More so, patients with BCL6 rearrangement were 5.5-fold more likely associated with extranodal involvement compared with their BCL2-rearranged peers. Moreover, ~60.0% of the BCL6-rearranged DH-HGBL cases were non-GCB, suggesting that including screening for BCL6 rearrangement in patients with the non-GCB phenotype may aid medical decision-making and therapeutic strategy. Contrary to contemporary data from western countries, 2 in every 3 patients with DH/TH-HGBL in Taiwan harbor BCL6 rearrangement. Consistent with present findings, we recommend mandatory screening for BCL6 rearrangement in patients with aggressive HGBL in Taiwan.

CKS1B overexpression implicates clinical aggressiveness of hepatocellular carcinomas but not p27(Kip1) protein turnover: an independent prognosticator with potential p27 (Kip1)-independent oncogenic attributes?

BACKGROUND: Through data mining the Stanford Microarray Database, the CKS1B transcript was found to be frequently upregulated in hepatocellular carcinomas (HCCs) with low alpha-fetal protein (AFP) expression. Together with SKP2, CKS1B is known to implicate p27(Kip1) protein turnover promoting cell-cycle progression. METHODS: CKS1B, p27(Kip1), and SKP2 were immunostained in 75 HCCs and correlated with clinicopathological features, local recurrence-free survival (LRFS), and overall survival (OS). Silencing of CKS1B and SKP2 with interference short-hairpin RNA (shRNA) was performed in SK-Hep1 and Hep-3B cell lines. RESULTS: Immunohistochemically, increased CKS1B and SKP2, and attenuated p27(Kip1) were all associated with tumor multiplicity (P < 0.05) and increasing American Joint Committee on Cancer (AJCC) stage (P < 0.05). Overexpression of CKS1B significantly correlated with advanced Okuda stages (P = 0.048) and SKP2 overexpression (P = 0.047). Neither CKS1B nor SKP2 was inversely related to p27(Kip1), which was reinforced by no alteration in p27(Kip1) abundance in HCC-derived cells with CKS1B or SKP2 silencing. Both CKS1B overexpression (P = 0.0011 and P = 0.0017) and p27(Kip1) attenuation (P = 0.0079 and P = 0.0085) were predictive of OS and LRFS, respectively, while SKP2 overexpression was associated with worse OS alone (P = 0.0043). Combined assessment of CKS1B and p27(Kip1) was able to robustly distinguish three prognostically different groups (P < 0.0001). In multivariate comparison, CKS1B overexpression represented the strongest independent adverse prognosticator [OS, P = 0.0235, hazard ratio (HR): 4.193; LRFS, P = 0.0204, HR: 4.262], followed by p27(Kip1) attenuation (OS, P = 0.0320, HR: 2.553; LRFS, P = 0.0262, HR: 2.533). CONCLUSIONS: CKS1B protein overexpression in HCCs is implicated in clinical aggressiveness but not in p27(Kip1) turnover, implying presence of p27(Kip1)-independent oncogenic attributes. The combined assessment of CKS1B and p27(Kip1) immunoexpressions effectively risk-stratifies HCCs with different prognoses, which may aid in the management of this deadly malignancy.

Icariin-mediated differentiation of mouse adipose-derived stem cells into cardiomyocytes.

In this study, we investigated the ability of mouse adipose-derived stem cells (ADSCs) to differentiate into a cardiac phenotype in vitro. Icariin (ICA) has previously been shown to induce cardiomyocyte (CM) differentiation of murine embryonic stem cells in vitro, but its effect on ADSCs remains unclear. We isolated ADSCs from white adipose tissue and analyzed selected surface antigens using flow cytometry. ADSCs and CMs were co-cultured in transwell plates, with or without the addition of either ICA or ICA plus the extracellular signal-regulated kinase (ERK) inhibitor PD98059. Cardiac-specific gene expression was examined by reverse transcription-polymerase chain reaction and western blotting. ICA facilitated differentiation of ADSCs into CMs that expressed cardiac-specific genes, including the transcription factors NKX-2.5, GATA-4, MLC-2v, α-actinin, and cardiac troponin-T. Expression of α-actinin, the Z band-constituting protein, was promoted by ICA in a dose- and time-dependent manner. ICA can induce ERK activation and cardiac-specific gene expression was partially inhibited by PD98059 after treatment with ICA. These results suggest that ICA-stimulated CM differentiation of ADSCs, and that it acted partially by activating ERK-dependent signaling pathways in vitro.

Evaluation of Cell-Penetrating Peptides Using Microfluidic In Vitro 3D Brain Endothelial Barrier.

In drug delivery to the human brain, blood vessels are a significant hurdle because they restrict the entry of most solutes to protect brain. To overcome this hurdle, an in vitro 3D model for brain endothelial barrier is developed using a microfluidic device with hydrogel providing a 3D extracellular matrix scaffold. Using the model, peptides known to utilize receptor-mediated transcytosis are verified, which has been one of the most promising mechanisms for brain-specific penetration. The cytotoxicity and cellular damage to the peptide are investigated and the receptor-mediated transcytosis and brain endothelial specific penetrating abilities of the peptides in a quantitative manner are demonstrated. As a preclinical test, applying the quantification assays conducted in this study are suggested, including the penetrating ability, cytotoxicity, endothelial damage, and receptor specificity. Using this microfluidic device as an in vitro platform for evaluating various brain targeting drugs and drug carrier candidates is also proposed.

Inactivation of MTOR promotes autophagy-mediated epithelial injury in particulate matter-induced airway inflammation.

Particulate matter (PM) is able to induce airway epithelial injury, while the detailed mechanisms remain unclear. Here we demonstrated that PM exposure inactivated MTOR (mechanistic target of rapamycin kinase), enhanced macroautophagy/autophagy, and impaired lysosomal activity in HBE (human bronchial epithelial) cells and in mouse airway epithelium. Genetic or pharmaceutical inhibition of MTOR significantly enhanced, while inhibition of autophagy attenuated, PM-induced IL6 expression in HBE cells. Consistently, club-cell-specific deletion of Mtor aggravated, whereas loss of Atg5 in bronchial epithelium reduced, PM-induced airway inflammation. Interestingly, the augmented inflammatory responses caused by MTOR deficiency were markedly attenuated by blockage of downstream autophagy both in vitro and in vivo. Mechanistically, the dysregulation of MTOR-autophagy signaling was partially dependent on activation of upstream TSC2, and interacted with the TLR4-MYD88 to orchestrate the downstream NFKB activity and to regulate the production of inflammatory cytokines in airway epithelium. Moreover, inhibition of autophagy reduced the expression of EPS15 and the subsequent endocytosis of PM. Taken together, the present study provides a mechanistic explanation for how airway epithelium localized MTOR-autophagy axis regulates PM-induced airway injury, suggesting that activation of MTOR and/or suppression of autophagy in local airway might be effective therapeutic strategies for PM-related airway disorders.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; ALI: air liquid interface; AP2: adaptor related protein complex 2; ATG: autophagy related; BALF: bronchoalveolar lavage fluid; COPD: chronic obstructive pulmonary disease; CXCL: C-X-C motif chemokine ligand; DOX: doxycycline; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EPS15: epidermal growth factor receptor pathway substrate 15; HBE: human bronchial epithelial; H&E: hematoxylin & eosin; IKK: IKB kinase; IL: interleukin; LAMP2: lysosomal-associated membrane protein 2; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTEC: mouse tracheal epithelial cells; MTOR: mechanistic target of rapamycin kinase; MYD88: MYD88 innate immune signal transduction adaptor; NFKB: nuclear factor of kappa B; NFKBIA: NFKB inhibitor alpha; PM: particulate matter; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RELA: RELA proto-oncogene, NFKB subunit; SCGB1A1: secretoglobin family 1A member 1; siRNA: small interfering RNAs; SQSTM1: sequestosome 1; TEM: transmission electronic microscopy; TLR4: toll like receptor 4; TSC2: TSC complex subunit 2.