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This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115-149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC 50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC 50 value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer types, indicating a poor survival prognosis with a more aggressive biology of metastasis to the lung and a short response duration to available therapies. Ibulocydine (IB) is a novel (cyclin-dependent kinase) CDK7/9 inhibitor prodrug displaying potent anti-cancer effects against various cancer cell types. We performed in vitro and in vivo experiments to determine whether IB inhibits metastasis and eventually overcomes the poor drug response in TNBC. The result showed that IB inhibited the growth of TNBC cells by inducing caspase-mediated apoptosis and blocking metastasis by reducing MMP-9 expression in vitro. Concurrently, in vivo experiments using the metastasis model showed that IB inhibited metastasis of MDA-MB-231-Luc cells to the lung. Collectively, these results demonstrate that IB inhibited the growth of TNBC cells and blocked metastasis by regulating MMP-9 expression, suggesting a novel therapeutic agent for metastatic TNBC.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC 50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

Ovarian aging is a major obstacle in assisted reproductive medicine because it leads to ovarian dysfunction in women of advanced age. Currently, there are no effective treatments to cure age-related ovarian dysfunction. In this study, we investigated the effect of MIT-001 on the function of aged ovaries. Young and old mice were utilized in this study. MIT-001 was intraperitoneally administered, and the number of follicles and oocytes was analyzed. Each group was then retrieved for RNA and protein isolation. Total RNA was subjected to mRNA next-generation sequencing. Protein extracts from ovarian lysates were used to evaluate various cytokine levels in the ovaries. MIT-001 enhanced follicles and the number of oocytes were compared with non-treated old mice. MIT-001 downregulated immune response-related transcripts and cytokines in the ovaries of old mice. MIT-001 modulates the immune complex responsible for generating inflammatory signals and has the potential to restore the function of old ovaries and improve female fertility.

Bromodomain-containing protein 4 (BRD4) is an intracellular protein that regulates expression of various cellular functions. This study investigated whether BRD4 inhibition can alter the immunomodulatory and antitumor effects of radiation therapy (RT). A murine breast cancer cell line was implanted into BALB/c mice. The dual-tumor model was used to evaluate the abscopal effects of RT. A total of 24 Gy was delivered and BRD4 inhibitor was injected intravenously. Tumor size was measured, and in vivo imaging was performed to evaluate tumor growth. Flow cytometry and immunohistochemistry were performed to examine immunologic changes upon treatment. The combination of BRD4 inhibitor and RT significantly suppressed tumor growth compared to RT alone. BRD4 inhibitor reduced the size of the unirradiated tumor, indicating that it may induce systemic immune responses. The expression of HIF-1α and PD-L1 in the tumor was significantly downregulated by the BRD4 inhibitor. The proportion of M1 tumor-associated macrophages (TAMs) increased, and the proportion of M2 TAMs decreased upon BRD4 inhibition. BRD4 inhibitor expanded CD4 + and CD8 + T cell populations in the tumor microenvironment. Additionally, splenic monocytic myeloid derived suppressor cells, which were increased by RT, were reduced upon the addition of BRD4 inhibitor. Therefore, the addition of BRD4 inhibitor significantly enhanced the systemic antitumor responses of local RT.

Trolox is a potent antioxidant and a water-soluble analog of vitamin E. It has been used in scientific studies to examine oxidative stress and its impact on biological systems. Trolox has been shown to have a neuroprotective effect against ischemia and IL-1β-mediated neurodegeneration. In this study, we investigated the potential protective mechanisms of Trolox against a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mouse model. Western blotting, immunofluorescence staining, and ROS/LPO assays were performed to investigate the role of trolox against neuroinflammation, the oxidative stress mediated by MPTP in the Parkinson's disease (PD) mouse model (wild-type mice (C57BL/6N), eight weeks old, average body weight 25-30 g). Our study showed that MPTP increased the expression of α-synuclein, decreased tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels in the striatum and substantia nigra pars compacta (SNpc), and impaired motor function. However, Trolox treatment significantly reversed these PD-like pathologies. Furthermore, Trolox treatment reduced oxidative stress by increasing the expression of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Lastly, Trolox treatment inhibited the activated astrocytes (GFAP) and microglia (Iba-1), also reducing phosphorylated nuclear factor-κB, (p-NF-κB) and tumor necrosis factor-alpha (TNF-α) in the PD mouse brain. Overall, our study demonstrated that Trolox may exert neuroprotection on dopaminergic neurons against MPTP-induced oxidative stress, neuroinflammation, motor dysfunction, and neurodegeneration.

We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.

The blood-brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer's disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB's structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies.

Frequent mutation of APC (90%) in advanced colorectal cancer (CRC) results in the simultaneous activation of Wnt/β-catenin and AKT signaling pathways, and the current therapeutic limitations of the AKT inhibitors for treating CRC patients are nuclear β-catenin-induced EMT and bypassing apoptosis. In this study, we discover that the combinatorial treatment of an AKT inhibitor and KY1022, a β-catenin destabilizer, effectively overcomes the current limitations of API-2, an AKT inhibitor, by reducing nuclear β-catenin. Taken together, we demonstrate that the simultaneous suppression of Wnt/β-catenin with the AKT signaling pathways is an ideal strategy for suppressing the AKT-inhibitor-mediated metastasis and for maximizing the therapeutic effects of AKT inhibitors.

In mice, zygotic genome activation (ZGA) occurs in two steps: minor ZGA at the one-cell stage and major ZGA at the two-cell stage. Regarding the regulation of gene transcription, minor ZGA is known to have unique features, including a transcriptionally permissive state of chromatin and insufficient splicing processes. The molecular characteristics may originate from extremely open chromatin states in the one-cell stage zygotes, yet the precise underlying mechanism has not been well studied. Recently, the R-loop, a triple-stranded nucleic acid structure of the DNA/RNA hybrid, has been implicated in gene transcription and DNA replication. Therefore, in the present study, we examined the changes in R-loop dynamics during mouse zygotic development, and its roles in zygotic transcription or DNA replication. Our analysis revealed that R-loops persist in the genome of metaphase II oocytes and preimplantation embryos from the zygote to the blastocyst stage. In particular, zygotic R-loop levels dynamically change as development proceeds, showing that R-loop levels decrease as pronucleus maturation occurs. Mechanistically, R-loop dynamics are likely linked to ZGA, as inhibition of either DNA replication or transcription at the time of minor ZGA decreases R-loop levels in the pronuclei of zygotes. However, the induction of DNA damage by treatment with anticancer agents, including cisplatin or doxorubicin, does not elicit genome-wide changes in zygotic R-loop levels. Therefore, our study suggests that R-loop formation is mechanistically associated with the regulation of mouse ZGA, especially minor ZGA, by modulating gene transcription and DNA replication., Competing Interests: The authors declare no conflict of interest.

The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/10 4 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.

Immune checkpoint inhibitors have been successful in a wide range of tumor types but still have limited efficacy in immunologically cold tumors, such as breast cancers. We hypothesized that the combination of agonistic anti-OX40 (α-OX40) co-stimulation, PD-1 blockade, and radiotherapy would improve the therapeutic efficacy of the immune checkpoint blockade in a syngeneic murine triple-negative breast cancer model. Murine triple-negative breast cancer cells (4T1) were grown in immune-competent BALB/c mice, and tumors were irradiated with 24 Gy in three fractions. PD-1 blockade and α-OX40 were administered five times every other day. Flow cytometric analyses and immunohistochemistry were used to monitor subsequent changes in the immune cell repertoire. The combination of α-OX40, radiotherapy, and PD-1 blockade significantly improved primary tumor control, abscopal effects, and long-term survival beyond 2 months (60%). In the tumor microenvironment, the ratio of CD8+ T cells to CD4 + FOXP3+ regulatory T cells was significantly elevated and exhausted CD8+ T cells (PD-1+, CTLA-4+, TIM-3+, or LAG-3+ cells) were significantly reduced in the triple combination group. Systemically, α-OX40 co-stimulation and radiation significantly increased the CD103+ dendritic cell response in the spleen and plasma IFN-γ, respectively. Together, our results suggest that the combination of α-OX40 co-stimulation and radiation is a viable approach to overcome therapeutic resistance to PD-1 blockade in immunologically cold tumors, such as triple-negative breast cancer.

Tyrosine-protein kinase (Syk) plays a potential role in neuroinflammation and adaptive immune responses in several neurodegenerative conditions. Seeing the significant role of Syk in the pathophysiology of neurodegeneration, several pharmacological inhibitors have been developed. One of the known inhibitors of Syk is BAY61-3606, which has shown efficacies in Alzheimer's disease (AD) through regulating amyloid production. However, little is known about its efficacies in neuroinflammation and neurodegeneration. Our finding showed that Syk expression was up-regulated by lipopolysaccharide (LPS)-dependent manner, and BAY61-3606 significantly suppressed the activated microglia (ionized calcium-binding adaptor molecule 1 [Iba-1]) and the inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin 1-beta [IL-1β], IL-6) and other inflammatory mediators (nuclear factor kappa B [NF-κB], cyclooxygenase-2 [Cox-2], and inducible nitric axide synthase [iNOS]) in the lipopolysaccharide (LPS)-treated in vivo and in vitro models. Moreover, BAY61-3606 significantly reduced microglia-mediated neuronal cell death by regulating the expression of Cytochrome C and Bim (B-cell lymphoma 2 [BCL-2] interacting mediator of cell death) in the LPS-treated mice brain and HT22 cells. Furthermore, the expression of synaptic markers, synaptosomal-associated protein, 25 kDa (SNAP25), synaptophysin (Syp), and postsynaptic density protein-95 (PSD95) in LPS-challenged mice showed that BAY61-3606 significantly recovered the synaptic markers. Finally, we have analyzed the effects of BAY61-3606 against memory and cognitive dysfunctions in the LPS injected mice. The Y-maze test and Passive avoidance test suggested that BAY61-3606 significantly protected against LPS-induced cognitive and memory dysfunctions. The current findings not only highlight the mechanisms of Syk in the pathophysiology of neuro-inflammation, but also support the therapeutic efficacy of BAY61-3606 in the management of neurodegeneration.

The purpose of this study is to evaluate the effect of the bioconversion products of Oenanthe javanica extract fermented by Lactiplantibacillus plantarum (OEFL) on relieving hangovers and improving liver function. In addition, the bioactive substance of the OEFL, which alleviates hangover and ethanol-induced liver damage, was identified and its bioactive property was verified through in vivo experiments. In major substances analysis using high-performance liquid chromatography, OEFL produced 9.5-fold higher p -coumaric acid than the O. Javanica extract (OE). In addition, considering that quinic acid, which is not present in the OE, was produced in the OEFL it was confirmed that chlorogenic acid was decomposed into quinic acid by bioconversion. In the in vivo experiment using Sprague-Dawley rats, the OEFL and p -coumaric acid diets reduced blood ethanol, acetaldehyde, GPT, and ALP concentrations, increasing blood albumin concentrations compared to ethanol-administered groups, demonstrating that OEFL and p -coumaric acid, the main substance in the OEFL, improved ethanol-induced liver damage. Furthermore, the OEFL and its main bioactive substance, p -coumaric acid, alleviated liver fibrosis by downregulating TGF-β, SMAD-2, SMAD-4, α-SMA , and upregulating MMP-1 . Therefore, OEFL is expected to be used as a functional food or pharmaceutical material as it has been confirmed to effectively relieve hangovers, prevent liver damage, and delay liver fibrosis in ethanol-induced liver damages.

The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.

Melanin is a brown or black pigment that protects skin from ultraviolet radiation and reactive oxygen species (ROS). However, overproduction of melanin is associated with lentigines, melasma, freckles and skin cancer. Licorice has shown antioxidant, anti-tumor, anti-platelet, anti-inflammatory and immunomodulatory activities and is used as a natural treatment for skin whitening. We aimed to confirm the potential of Wongam, a new cultivar of licorice developed by the Rural Development Administration (RDA), as a whitening agent in cosmetics. In addition, we verified the effect of heat treatment on the bioactivity of licorice by comparing antioxidant and anti-melanogenic activities of licorice extract before and after heating (130 °C). The heat-treated licorice extract (WH-130) showed higher radical-scavenging activities in the ABTS + (2,2'-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays. In addition, WH-130 inhibited melanogenesis more effectively due to downregulation of tyrosinase in B16F10 melanoma cells than non-heated licorice extract. Moreover, heat treatment increased total phenolic content. In particular, isoliquiritigenin, an antioxidant and anti-melanogenic compound of licorice, was produced by heat treatment. In conclusion, WH-130, with increased levels of bioactive phenolics such as isoliquiritigenin, has potential for development into a novel skin whitening material with applications in cosmetics.

Cellular immunotherapy has recently emerged as a fourth pillar in cancer treatment co-joining surgery, chemotherapy and radiotherapy. Where, the discovery of immune checkpoint blockage or inhibition (ICB/ICI), anti-PD-1/PD-L1 and anti-CTLA4-based, therapy has revolutionized the class of cancer treatment at a different level. However, some cancer patients escape this immune surveillance mechanism and become resistant to ICB-therapy. Therefore, a more advanced or an alternative treatment is required urgently. Despite the functional importance of epitranscriptomics in diverse clinico-biological practices, its role in improving the efficacy of ICB therapeutics has been limited. Consequently, our study encapsulates the evidence, as a possible strategy, to improve the efficacy of ICB-therapy by co-targeting molecular checkpoints especially N 6 A-modification machineries which can be reformed into RNA modifying drugs (RMD). Here, we have explained the mechanism of individual RNA-modifiers (editor/writer, eraser/remover, and effector/reader) in overcoming the issues associated with high-dose antibody toxicities and drug-resistance. Moreover, we have shed light on the importance of suppressor of cytokine signaling (SOCS/CISH) and microRNAs in improving the efficacy of ICB-therapy, with brief insight on the current monoclonal antibodies undergoing clinical trials or already approved against several solid tumor and metastatic cancers. We anticipate our investigation will encourage researchers and clinicians to further strengthen the efficacy of ICB-therapeutics by considering the importance of epitranscriptomics as a personalized medicine.

The intended scapular motion is a strategy to strengthen the lower trapezius (LT). However, few studies have explored the effects of the intended scapular posterior tilt motion on selective LT activation. Thus, the present study investigated the effect of the intended scapular posterior tilt on the electromyography (EMG) activity of trapezius muscles during prone shoulder horizontal abduction (PSHA). Eighteen asymptomatic men performed three types of PSHA: (1) preferred PSHA, (2) PSHA with the intended scapular posterior tilt, and (3) PSHA with the intended scapular posterior tilt and trunk extension. EMG activity of the upper trapezius (UT), middle trapezius (MT), and LT were measured during PSHAs. Scapular posterior tilt angle, with and without the intended scapular posterior tilt, were measured using inclinometer. The results indicated that LT muscle activity increased when scapular posterior tilt was applied with and without trunk extension (14-16%), compared to the preferred condition, during PSHA ( p < 0.05). However, the addition of trunk extension to PSHA with the intended scapular posterior tilt increased the UT muscle activity (28%) and the UT/LT (29%) and UT/MT (31%) ratios ( p < 0.05). The scapular posterior tilt angle was higher (15%) when applying the intended scapular posterior tilt ( p = 0.020). These findings suggest that the intended scapular posterior tilt may be a useful strategy for selective LT muscle activation.

Breast cancer is the most commonly occurring cancer in women of Western countries and is the leading cause of cancer-related mortality. The breast tumor microenvironment contains immune cells, fibroblasts, adipocytes, mesenchymal stem cells, and extracellular matrix. Among these cells, macrophages or tumor-associated macrophages (TAMs) are the major components of the breast cancer microenvironment. TAMs facilitate metastasis of the breast tumor and are responsible for poor clinical outcomes. High TAM density was also found liable for the poor prognosis of breast cancer. These observations make altering TAM function a potential therapeutic target to treat breast cancer. The present review summarizes the origin of TAMs, mechanisms of macrophage recruitment and polarization in the tumor, and the contributions of TAMs in tumor progression. We have also discussed our current knowledge about TAM-targeted therapies and the roles of miRNAs and exosomes in re-educating TAM function.

The use of NF 3 is significantly increasing every year. However, NF 3 is a greenhouse gas with a very high global warming potential. Therefore, the development of a material to replace NF 3 is required. F 3 NO is considered a potential replacement to NF 3 . In this study, the characteristics and cleaning performance of the F 3 NO plasma to replace the greenhouse gas NF 3 were examined. Etching of SiO 2 thin films was performed, the DC offset of the plasma of both gases (i.e., NF 3 and F 3 NO) was analyzed, and a residual gas analysis was performed. Based on the analysis results, the characteristics of the F 3 NO plasma were studied, and the SiO 2 etch rates of the NF 3 and F 3 NO plasmas were compared. The results show that the etch rates of the two gases have a difference of 95% on average, and therefore, the cleaning performance of the F 3 NO plasma was demonstrated, and the potential benefit of replacing NF 3 with F 3 NO was confirmed.

With growing interest in healthcare, wearable healthcare devices have been developed and researched. In particular, near-field communication (NFC) based wearable devices have been actively studied for device miniaturization. Herein, this article proposes a low-cost and convenient healthcare system, which can monitor heart rate and temperature using a wireless/battery-free sensor and the customized smartphone application. The authors designed and fabricated a customized healthcare device based on the NFC system, and developed a smartphone application for real-time data acquisition and processing. In order to achieve compact size without performance degradation, a dual-layered layout is applied to the device. The authors demonstrate that the device can operate as attached on various body sites such as wrist, fingertip, temple, and neck due to outstanding flexibility of device and adhesive strength between the device and the skin. In addition, the data processing flow and processing result are presented for offering heart rate and skin temperature. Therefore, this work provides an affordable and practical pathway for the popularization of wireless wearable healthcare system. Moreover, the proposed platform can easily delivery the measured health information to experts for contactless/personal health consultation.

Epithelial to mesenchymal transition (EMT) is associated with resistance during EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. Here, we investigated whether EMT is associated with acquired resistance to 3rd generation EGFR-TKIs, and we explored the effects of cyclin-dependent kinase 7 (CDK7) inhibitors on EMT-mediated EGFR-TKIs resistance in non-small cell lung cancer (NSCLC). We established 3rd generation EGFR-TKI resistant cell lines (H1975/WR and H1975/OR) via repeated exposure to WZ4002 and osimertinib. The two resistant cell lines showed phenotypic changes to a spindle-cell shape, had a reduction of epithelial marker proteins, an induction of vimentin expression, and enhanced cellular mobility. The EMT-related resistant cells had higher sensitivity to THZ1 than the parental cells, although THZ1 treatment did not inhibit EGFR activity. This phenomenon was also observed in TGF-β1 induced EMT cell lines. THZ1 treatment induced G2/M cell cycle arrest and apoptosis in all of the cell lines. In addition, THZ1 treatment led to drug-tolerant, EMT-related resistant cells, and these THZ1-tolerant cells partially recovered their sensitivity to 3rd generation EGFR-TKIs. Taken together, EMT was associated with acquired resistance to 3rd generation EGFR-TKIs, and CDK7 inhibitors could potentially be used as a therapeutic strategy to overcome EMT associated EGFR-TKI resistance in NSCLC.

Poly(L-lactic) acid (PLLA) is among the most promising polymers for bone fixation, repair, and tissue engineering due to its biodegradability and relatively good mechanical strength. Despite these beneficial characteristics, its poor bioactivity often requires incorporation of bioactive ceramic materials. A bioresorbable composite made of PLLA and hydroxyapatite (HA) may improve biocompatibility but typically causes deterioration in mechanical properties, and bioactive coatings inevitably carry a risk of coating delamination. Therefore, in this study, we embedded micropatterned HA on the surface of PLLA to improve bioactivity while eliminating the risk of HA delamination. An HA pattern was successfully embedded in a PLLA matrix without degeneration of the matrix's mechanical properties, thanks to a transfer technique involving conversion of Mg to HA. Furthermore, patterned HA/PLLA's biological response outperformed that of pure PLLA. These results confirm patterned HA/PLLA as a candidate for wide acceptance in biodegradable load-bearing implant applications.

In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure-function relationships of G protein-coupled receptors during signal transduction.

Neuroblastoma is the most common extracranial solid tumor in childhood. The different treatments available for neuroblastoma are challenged by high rates of resistance, recurrence, and progression, most notably in advanced cases and highly malignant tumors. Therefore, the development of more targeted therapies, which are biocompatible and without undesired side effects, is highly desirable. The mechanisms of actions of platinum nanoparticles (PtNPs) and retinoic acid (RA) in neuroblastoma have remained unclear. In this study, the anticancer effects of PtNPs and RA on neuroblastoma were assessed. We demonstrated that treatment of SH-SY5Y cells with the combination of PtNPs and RA resulted in improved anticancer effects. The anticancer effects of the two compounds were mediated by cytotoxicity, oxidative stress (OS), mitochondrial dysfunction, endoplasmic reticulum stress (ERS), and apoptosis-associated networks. Cytotoxicity was confirmed by leakage of lactate dehydrogenase (LDH) and intracellular protease, and oxidative stress increased the level of reactive oxygen species (ROS), 4-hydroxynonenal (HNE), malondialdehyde (MDA), and nitric oxide (NO), and protein carbonyl content (PCC). The combination of PtNPs and RA caused mitochondrial dysfunction by decreasing the mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) content, number of mitochondria, and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Endoplasmic reticulum-mediated stress and apoptosis were confirmed by upregulation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4), p53, Bax, and caspase-3 and down regulation of B-cell lymphoma 2 (BCl-2). PtNPs and RA induced apoptosis, and oxidative DNA damage was evident by the accumulation of 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG). Finally, PtNPs and RA increased the differentiation and expression of differentiation markers. Differentiated SH-SY5Y cells pre-treated with PtNPs or RA or the combination of both were more sensitive to the cytotoxic effect of cisplatin than undifferentiated cells. To our knowledge, this is the first study to demonstrate the effect of the combination of PtNPs and RA in neuroblastoma cells. PtNPs may be a potential preconditioning or adjuvant compound in chemotherapeutic treatment. The results of this study provide a rationale for clinical evaluation of the combination of PtNPs and RA for the treatment of children suffering from high-risk neuroblastoma.

Melatonin (MLT) is a powerful chronobiotic hormone that controls a multitude of circadian rhythms at several levels and, in recent times, has garnered considerable attention both from academia and industry. In several studies, MLT has been discussed as a potent neuroprotectant, anti-apoptotic, anti-inflammatory, and antioxidative agent with no serious undesired side effects. These characteristics raise hopes that it could be used in humans for central nervous system (CNS)-related disorders. MLT is mainly secreted in the mammalian pineal gland during the dark phase, and it is associated with circadian rhythms. However, the production of MLT is not only restricted to the pineal gland; it also occurs in the retina, Harderian glands, gut, ovary, testes, bone marrow, and lens. Although most studies are limited to investigating the role of MLT in the CNS and related disorders, we explored a considerable amount of the existing literature. The objectives of this comprehensive review were to evaluate the impact of MLT on the CNS from the published literature, specifically to address the biological functions and potential mechanism of action of MLT in the CNS. We document the effectiveness of MLT in various animal models of brain injury and its curative effects in humans. Furthermore, this review discusses the synthesis, biology, function, and role of MLT in brain damage, and as a neuroprotective, antioxidative, anti-inflammatory, and anticancer agent through a collection of experimental evidence. Finally, it focuses on the effect of MLT on several neurological diseases, particularly CNS-related injuries., Competing Interests: The authors declare no conflict of interest.

An accurate and reliable method based on ion trap-time of flight mass spectrometry (IT-TOF MS) was developed for screening phosphodiesterase-5 inhibitors, including sildenafil, vardenafil, and tadalafil, and their analogs in dietary supplements. Various parameters affecting liquid chromatographic separation and IT-TOF detection were investigated, and the optimal conditions were determined. The separation was achieved on a reversed-phase column under gradient elution using acetonitrile and water containing 0.2% acetic acid at a flow rate of 0.2 mL/min. The chromatographic eluents were directly ionized in the IT-TOF system equipped with an electrospray ion source operating in the positive ion mode. The proposed screening method was validated by assessing its linearity, precision, and accuracy. Sequential tandem MS was conducted to obtain structural information of the references, and the fragmentation mechanism of each reference was proposed for providing spectral insight for newly synthesized analogs. Structural information, including accurate masses of both parent and fragment ions, was incorporated into the MS n spectral library. The developed method was successfully applied for screening adulterated dietary supplement samples.

The extensive usage of silver nanoparticles (AgNPs) as medical products such as antimicrobial and anticancer agents has raised concerns about their harmful effects on human beings. AgNPs can potentially induce oxidative stress and apoptosis in cells. However, humanin (HN) is a small secreted peptide that has cytoprotective and neuroprotective cellular effects. The aim of this study was to assess the harmful effects of AgNPs on human neuroblastoma SH-SY5Y cells and also to investigate the protective effect of HN from AgNPs-induced cell death, mitochondrial dysfunctions, DNA damage, and apoptosis. AgNPs were prepared with an average size of 18 nm diameter to study their interaction with SH-SY5Y cells. AgNPs caused a dose-dependent decrease of cell viability and proliferation, induced loss of plasma-membrane integrity, oxidative stress, loss of mitochondrial membrane potential (MMP), and loss of ATP content, amongst other effects. Pretreatment or co-treatment of HN with AgNPs protected cells from several of these AgNPs induced adverse effects. Thus, this study demonstrated for the first time that HN protected neuroblastoma cells against AgNPs-induced neurotoxicity. The mechanisms of the HN-mediated protective effect on neuroblastoma cells may provide further insights for the development of novel therapeutic agents against neurodegenerative diseases., Competing Interests: The authors declare no conflict of interest.

Many Salicaceae s.l. plants are recognized for their important role in the production of products such as wood, oils, and medicines, and as a model organism in life studies. However, the difference in plastid sequence, phylogenetic relationships, and lineage diversification of the family Salicaceae s.l. remain poorly understood. In this study, we compare 24 species representing 18 genera of the family. Simple sequence repeats (SSRs) are considered effective molecular markers for plant species identification and population genetics. Among them, a total of 1798 SSRs were identified, among which mononucleotide repeat was the most common with 1455 accounts representing 80.92% of the total. Most of the SSRs are located in the non-coding region. We also identified five other types of repeats, including 1750 tandems, 434 forward, 407 palindromic, 86 reverse, and 30 complementary repeats. The species in Salicaceae s.l. have a conserved plastid genome. Each plastome presented a typical quadripartite structure and varied in size due to the expansion and contraction of the inverted repeat (IR) boundary, lacking major structural variations, but we identified six divergence hotspot regions. We obtained phylogenetic relationships of 18 genera in Salicaceae s.l. and the 24 species formed a highly supported lineage. Casearia was identified as the basal clade. The divergence time between Salicaceae s.l. and the outgroup was estimated as ~93 Mya; Salix , and Populus diverged around 34 Mya, consistent with the previously reported time. Our research will contribute to a better understanding of the phylogenetic relationships among the members of the Salicaceae s.l., Competing Interests: The authors declare no conflicts of interest.

Generally, platinum nanoparticles (PtNPs) are considered non-toxic; however, toxicity depends on the size, dose, and physico-chemical properties of materials. Owing to unique physico-chemical properties, PtNPs have emerged as a material of interest for several biomedical applications, particularly therapeutics. The adverse effect of PtNPs on the human monocytic cell line (THP-1) is not well-established and remains elusive. Exposure to PtNPs may trigger oxidative stress and eventually lead to inflammation. To further understand the toxicological properties of PtNPs, we studied the effect of biologically synthesized ultra-small PtNPs on cytotoxicity, genotoxicity, and proinflammatory responses in the human monocytic cell line (THP-1). Our observations clearly indicated that PtNPs induce cytotoxicity in a dose-dependent manner by reducing cell viability and proliferation. The cytotoxicity of THP-1 cells correlated with an increase in the leakage of lactate dehydrogenase, generation of reactive oxygen species, and production of malondialdehyde, nitric oxide, and carbonylated proteins. The involvement of mitochondria in cytotoxicity and genotoxicity was confirmed by loss of mitochondrial membrane potential, lower ATP level, and upregulation of proapoptotic and downregulation of antiapoptotic genes. Decreases in the levels of antioxidants such as reduced glutathione (GSH), oxidized glutathione (GSH: GSSG), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and thioredoxin (TRX) were indicative of oxidative stress. Apoptosis was confirmed with the significant upregulation of key apoptosis-regulating genes. Oxidative DNA damage was confirmed by the increase in the levels of 8-oxodG and 8-oxoG and upregulation of DNA damage and repair genes. Finally, the proinflammatory responses to PtNPs was determined by assessing the levels of multiple cytokines such as interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein 1 (MCP-1). All the cytokines were significantly upregulated in a dose-dependent manner. Collectively, these observations suggest that THP-1 cells were vulnerable to biologically synthesized ultra-small PtNPs., Competing Interests: The authors declare no conflicts of interest.

Metal nanoparticles and the combination of metal nanoparticles with graphene oxide are widely used in environmental, agriculture, textile, and therapeutic applications. The effect of graphene oxide-green platinum nanoparticles (GO-PtNPs) on human prostate cancer cells (LNCaP) is unclear. Therefore, this study aimed to synthesize a nanocomposite of GO-PtNPs and evaluate their effect on prostate cancer cells. Herein, we synthesized GO-PtNPs using vanillin and characterized GO-PtNPs. GO-PtNP cytotoxicity in LNCaP cells was demonstrated by measuring cell viability and proliferation. Both decreased in a dose-dependent manner compared to that by GO or PtNPs alone. GO-PtNP cytotoxicity was confirmed by increased lactate dehydrogenase release and membrane integrity loss. Oxidative stress induced by GO-PtNPs increased malondialdehyde, nitric oxide, and protein carbonyl contents. The effective reactive oxygen species generation impaired the cellular redox balance and eventually impaired mitochondria by decreasing the membrane potential and ATP level. The cytotoxicity to LNCaP cells was correlated with increased expression of proapoptotic genes (p53, p21, Bax, Bak, caspase 9, and caspase 3) and decreased levels of antiapoptotic genes (Bcl2 and Bcl-xl). Activation of the key regulators p53 and p21 inhibited the cyclin-dependent kinases Cdk2 and Cdk4, suggesting that p53 and p21 activation in GO-PtNP-treated cells caused genotoxic stress and apoptosis. The increased expression of genes involved in cell cycle arrest and DNA damage and repair, and increased levels of 8-oxo-deoxyguanosine and 8-oxoguanine suggested that GO-PtNPs potentially induce oxidative damage to DNA. Thus, GO-PtNPs are both cytotoxic and genotoxic. LNCaP cells appear to be more susceptible to GO-PtNPs than to GO or PtNPs. Therefore, GO-PtNPs have potential as an alternate and effective cancer therapeutic agent. Finally, this work shows that the combination of graphene oxide with platinum nanoparticles opens new perspectives in cancer therapy. However further detailed mechanistic studies are required to elucidate the molecular mechanism of GO-PtNPs induced cytotoxicity in prostate cancer.

Exosomes are extracellular vesicles that contain a specific composition of proteins, lipids, RNA, and DNA. They are derived from endocytic membranes and can transfer signals to recipient cells, thus mediating a novel mechanism of cell-to-cell communication. They are also thought to be involved in cellular waste disposal. Exosomes play significant roles in various biological functions, including the transfer of biomolecules such as RNA, proteins, enzymes, and lipids and the regulation of numerous physiological and pathological processes in various diseases. Because of these properties, they are considered to be promising biomarkers for the diagnosis and prognosis of various diseases and may contribute to the development of minimally invasive diagnostics and next generation therapies. The biocompatible nature of exosomes could enhance the stability and efficacy of imaging probes and therapeutics. Due to their potential use in clinical applications, exosomes have attracted much research attention on their roles in health and disease. To explore the use of exosomes in the biomedical arena, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are well-understood. Herein, we discuss the history, biogenesis, release, isolation, characterization, and biological functions of exosomes, as well as the factors influencing their biogenesis and their technical and biological challenges. We conclude this review with a discussion on the future perspectives of exosomes., Competing Interests: The authors declare no conflict of interest.

Graphene and its derivatives are emerging as attractive materials for biomedical applications, including antibacterial, gene delivery, contrast imaging, and anticancer therapy applications. It is of fundamental importance to study the cytotoxicity and biocompatibility of these materials as well as how they interact with the immune system. The present study was conducted to assess the immunotoxicity of graphene oxide (GO) and vanillin-functionalized GO (V-rGO) on THP-1 cells, a human acute monocytic leukemia cell line. The synthesized GO and V-rGO were characterized by using various analytical techniques. Various concentrations of GO and V-rGO showed toxic effects on THP-1 cells such as the loss of cell viability and proliferation in a dose-dependent manner. Cytotoxicity was further demonstrated as an increased level of lactate dehydrogenase (LDH), loss of mitochondrial membrane potential (MMP), decreased level of ATP content, and cell death. Increased levels of reactive oxygen species (ROS) and lipid peroxidation caused redox imbalance in THP-1 cells, leading to increased levels of malondialdehyde (MDA) and decreased levels of anti-oxidants such as glutathione (GSH), glutathione peroxidase (GPX), super oxide dismutase (SOD), and catalase (CAT). Increased generation of ROS and reduced MMP with simultaneous increases in the expression of pro-apoptotic genes and downregulation of anti-apoptotic genes suggest that the mitochondria-mediated pathway is involved in GO and V-rGO-induced apoptosis. Apoptosis was induced consistently with the significant DNA damage caused by increased levels of 8-oxo-dG and upregulation of various key DNA-regulating genes in THP-1 cells, indicating that GO and V-rGO induce cell death through oxidative stress. As a result of these events, GO and V-rGO stimulated the secretion of various cytokines and chemokines, indicating that the graphene materials induced potent inflammatory responses to THP-1 cells. The harshness of V-rGO in all assays tested occurred because of better charge transfer, various carbon to oxygen ratios, and chemical compositions in the rGO. Overall, these findings suggest that it is essential to better understand the parameters governing GO and functionalized GO in immunotoxicity and inflammation. Rational design of safe GO-based formulations for various applications, including nanomedicine, may result in the development of risk management methods for people exposed to graphene and graphene family materials, as these nanoparticles can be used as delivery agents in various biomedical applications.

Cancer represents a group of heterogeneous diseases characterized by uncontrolledgrowth and spread of abnormal cells, ultimately leading to death. Nanomedicine plays a significantrole in the development of nanodrugs, nanodevices, drug delivery systems and nanocarriers. Someof the major issues in the treatment of cancer are multidrug resistance (MDR), narrow therapeuticwindow and undesired side effects of available anticancer drugs and the limitations of anticancerdrugs. Several nanosystems being utilized for detection, diagnosis and treatment such as theranosticcarriers, liposomes, carbon nanotubes, quantum dots, polymeric micelles, dendrimers and metallicnanoparticles. However, nonbiodegradable nanoparticles causes high tissue accumulation andleads to toxicity. MDR is considered a major impediment to cancer treatment due to metastatictumors that develop resistance to chemotherapy. MDR contributes to the failure of chemotherapiesin various cancers, including breast, ovarian, lung, gastrointestinal and hematological malignancies.Moreover, the therapeutic efficiency of anticancer drugs or nanoparticles (NPs) used alone is lessthan that of the combination of NPs and anticancer drugs. Combination therapy has long beenadopted as the standard first-line treatment of several malignancies to improve the clinical outcome.Combination therapy with anticancer drugs has been shown to generally induce synergistic drugactions and deter the onset of drug resistance. Therefore, this review is designed to report andanalyze the recent progress made to address combination therapy using NPs and anticancer drugs.We first provide a comprehensive overview of the angiogenesis and of the different types of NPscurrently used in treatments of cancer; those emphasized in this review are liposomes, polymericNPs, polymeric micelles (PMs), dendrimers, carbon NPs, nanodiamond (ND), fullerenes, carbonnanotubes (CNTs), graphene oxide (GO), GO nanocomposites and metallic NPs used forcombination therapy with various anticancer agents. Nanotechnology has provided the convenienttools for combination therapy. However, for clinical translation, we need continued improvementsin the field of nanotechnology., Competing Interests: The authors declare no competing financial interests.

Although many treatment strategies have been reported for lung disease, the mechanism of combination therapy using silver nanoparticles (AgNPs) and histone deacetylases inhibitors (HDACi) remains unclear. Therefore, innovative treatment strategies are essential for addressing the therapeutic challenges of this highly aggressive lung cancer. AgNPs and HDACi seem to be the best candidates for anticancer therapy because of their anti-proliferative effect in a variety of cancer cells. First, we synthesized AgNPs using wogonin as a reducing and stabilizing agent, following which the synthesized AgNPs were characterized by various analytical techniques. The synthesized AgNPs exhibited dose- and size-dependent toxicity towards A549 cells. Interestingly, the combination of AgNPs and MS-275 significantly induces apoptosis, which was accompanied by an increased level of reactive oxygen species (ROS); leakage of lactate dehydrogenase (LDH); secretion of TNFα; dysfunction of mitochondria; accumulation autophagosomes; caspase 9/3 activation; up and down regulation of pro-apoptotic genes and anti-apoptotic genes, respectively; and eventually, induced DNA-fragmentation. Our findings suggest that AgNPs and MS-275 induce cell death in A549 lung cells via the mitochondrial-mediated intrinsic apoptotic pathway. Finally, our data show that the combination of AgNPs and MS-275 is a promising new approach for the treatment of lung cancer and our findings contribute to understanding the potential roles of AgNPs and MS-275 in pulmonary disease. However, further study is warranted to potentiate the use of this combination therapy in cancer therapy trials.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice (Oryza sativa L.). In this study, we investigated the genome-wide transcription patterns of two Xoo strains (KACC10331 and HB1009), which showed different virulence patterns against eight rice cultivars, including IRBB21 (carrying Xa21). In total, 743 genes showed a significant change (p-value < 0.001 in t-tests) in their mRNA expression levels in the HB1009 (K3a race) strain compared with the Xoo KACC10331 strain (K1 race). Among them, four remarkably enriched GO terms, DNA binding, transposition, cellular nitrogen compound metabolic process, and cellular macromolecule metabolic process, were identified in the upregulated genes. In addition, the expression of 44 genes was considerably higher (log2 fold changes > 2) in the HB1009 (K3a race) strain than in the Xoo KACC10331 (K1 race) strain. Furthermore, 13 and 12 genes involved in hypersensitive response and pathogenicity (hrp) and two-component regulatory systems (TCSs), respectively, were upregulated in the HB1009 (K3a race) strain compared with the Xoo KACC10331 (K1 race) strain, which we determined using either quantitative real-time PCR analysis or next-generation RNA sequencing. These results will be helpful to improve our understanding of Xoo and to gain a better insight into the Xoo-rice interactions.

To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) are well established surface techniques that provide both elemental and organic information from several monolayers of a sample surface, while also allowing depth profiling or image mapping to be carried out. The static TOF-SIMS with improved performances has expanded the application of TOF-SIMS to the study of a variety of organic, polymeric and biological materials. In this work, TOF-SIMS, XPS and Fourier Transform Infrared (FTIR) measurements were used to characterize commercial natural dyes and traditional silk fabric dyed with plant extracts dyes avoiding the time-consuming and destructive extraction procedures necessary for the spectrophotometric and chromatographic methods previously used. Silk textiles dyed with plant extracts were then analyzed for chemical and functional group identification of their dye components and mordants. TOF-SIMS spectra for the dyed silk fabric showed element ions from metallic mordants, specific fragment ions and molecular ions from plant-extracted dyes. The results of TOF-SIMS, XPS and FTIR are very useful as a reference database for comparison with data about traditional Korean silk fabric and to provide an understanding of traditional dyeing materials. Therefore, this study shows that surface techniques are useful for micro-destructive analysis of plant-extracted dyes and Korean dyed silk fabric.