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Dihydroflavonol 4-reductase (DFR) significantly influences the modification of flower color. To explore the role of DFR in the synthesis of strawberry anthocyanins, in this study, we downloaded the CDS sequences of the DFR gene family from the Arabidopsis genome database TAIR; the DFR family of forest strawberry was compared; then, a functional domain screen was performed using NCBI; the selected strawberry DFR genes were analyzed; and the expression characteristics of the family members were studied by qRT-PCR. The results showed that there are 57 members of the DFR gene family in strawberry, which are mainly expressed in the cytoplasm and chloroplast; most of them are hydrophilic proteins; and the secondary structure of the protein is mainly composed of α-helices and random coils. The analysis revealed that FvDFR genes mostly contain light, hormone, abiotic stress, and meristem response elements. From the results of the qRT-PCR analysis, the relative expression of each member of the FvDFR gene was significantly different, which was expressed throughout the process of fruit coloring. Most genes had the highest expression levels in the full coloring stage (S4). The expression of FvDFR30, FvDFR54, and FvDFR56 during the S4 period was 8, 2.4, and 2.4 times higher than during the S1 period, indicating that the DFR gene plays a key role in regulating the fruit coloration of strawberry. In the strawberry genome, 57 members of the strawberry DFR gene family were identified. The higher the DFR gene expression, the higher the anthocyanin content, and the DFR gene may be the key gene in anthocyanin synthesis. Collectively, the DFR gene is closely related to fruit coloring, which lays a foundation for further exploring the function of the DFR gene family.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose significant global health challenges. The results demonstrated that GB-2 at 200 μg/mL effectively increased the population of 293T-ACE2 cells with low RBD binding for both SARS-CoV-2 Omicron EG.5.1 and HV.1 variants by dual-color flow cytometry, indicating its ability to inhibit virus attachment. Further investigation revealed that (+)-catechin at 25 and 50 μg/mL did not significantly alter the ACE2–RBD interaction for the EG.5.1 variant. In contrast, theaflavin showed inhibitory effects at both 25 and 50 μg/mL for EG.5.1, while only the higher concentration was effective for HV.1. Notably, theaflavin 3-gallate exhibited a potent inhibition of ACE2–RBD binding for both variants at both concentrations tested. Molecular docking studies provided insight into the binding mechanisms of theaflavin and theaflavin 3-gallate with the RBD of EG.5.1 and HV.1 variants. Both compounds showed favorable docking scores, with theaflavin 3-gallate demonstrating slightly lower scores (−8 kcal/mol) compared to theaflavin (−7 kcal/mol) for both variants. These results suggest stable interactions between the compounds and key residues in the RBD, potentially explaining their inhibitory effects on virus attachment. In conclusion, GB-2, theaflavin, and theaflavin 3-gallate demonstrate significant potential as inhibitors of the ACE2–RBD interaction in Omicron variants, highlighting their therapeutic promise against COVID-19. However, these findings are primarily based on computational and in vitro studies, necessitating further in vivo research and clinical trials to confirm their efficacy and safety in humans.

Nipah virus (NiV) is known to be a highly pathogenic zoonotic virus, which is included in the World Health Organization Research & Development Blueprint list of priority diseases with up to 70% mortality rate. Due to its high pathogenicity and outbreak potency, a therapeutic countermeasure against NiV is urgently needed. As NiV needs to be handled within a Biological Safety Level (BSL) 4 facility, we had developed a safe drug screening platform utilizing a baculovirus expression vector system (BEVS) based on a NiV-induced syncytium formation that could be handled within a BSL-1 facility. To reconstruct the NiV-induced syncytium formation in BEVS, two baculoviruses were generated to express recombinant proteins that are responsible for inducing the syncytium formation, including one baculovirus exhibiting co-expressed NiV fusion protein (NiV-F) and NiV attachment glycoprotein (NiV-G) and another exhibiting human EphrinB2 protein. Interestingly, syncytium formation was observed in infected insect cells when the medium was modified to have a lower pH level and supplemented with cholesterol. Fusion inhibitory properties of several compounds, such as phytochemicals and a polysulfonated naphthylamine compound, were evaluated using this platform. Among these compounds, suramin showed the highest fusion inhibitory activity against NiV-induced syncytium in the baculovirus expression system. Moreover, our in silico results provide a molecular-level glimpse of suramin’s interaction with NiV-G’s central hole and EphrinB2’s G-H loop, which could be the possible reason for its fusion inhibitory activity.

Atopic dermatitis (AD) is a common skin disease worldwide. The major causes of AD are skin barrier defects, immune dysfunction, and oxidative stress. In this study, we investigated the anti-oxidation and anti-inflammation effects of Coffea arabica extract (CAE) and its regulation of the skin barrier and immune functions in AD. In vitro experiments revealed that CAE decreased the reactive oxygen species levels and inhibited the translocation of nuclear factor-κB (NF-κB), further reducing the secretion of interleukin (IL)-1β and IL-6 induced by interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α). Moreover, CAE decreased IFN-γ/TNF-α-induced NLR family pyrin domain-containing 3 (NLRP3), caspase-1, high-mobility group box 1 (HMGB1), and receptor for advanced glycation end products (RAGE) expression levels. It also restored the protein levels of skin barrier function-related markers including filaggrin and claudin-1. In vivo experiments revealed that CAE not only reduced the redness of the backs of mice caused by 2,4-dinitrochlorobenzene (DNCB) but also reduced the levels of pro-inflammatory factors in their skin. CAE also reduced transepidermal water loss (TEWL) and immune cell infiltration in DNCB-treated mice. Overall, CAE exerted anti-oxidation and anti-inflammation effects and ameliorated skin barrier dysfunction, suggesting its potential as an active ingredient for AD treatment.

Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson’s disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of BMAL1 and SIRT1 in the 6-OHDA-treated SH-SY5Y cells. Additionally, the SIRT1 inhibitor attenuated the effect of DT on BMAL1 and reduced the cell viability. The DT and SIRT1 activators activated SIRT1 and BMAL1, and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed.

Doxorubicin (DOX) as a chemotherapeutic agent can cause mitochondrial dysfunction and heart failure. COX5A has been described as an important regulator of mitochondrial energy metabolism. We investigate the roles of COX5A in DOX-induced cardiomyopathy and explore the underlying mechanisms. C57BL/6J mice and H9c2 cardiomyoblasts were treated with DOX, and the COX5A expression was assessed. An adeno-associated virus serum type 9 (AAV9) and lenti-virus system were used to upregulate COX5A expression. Echocardiographic parameters, morphological and histological analyses, transmission electron microscope and immunofluorescence assays were used to assess cardiac and mitochondrial function. In a human study, we found that cardiac COX5A expression was dramatically decreased in patients with end-stage dilated cardiomyopathy (DCM) compared to the control group. COX5A was significantly downregulated following DOX stimulation in the heart of mice and H9c2 cells. Reduced cardiac function, decreased myocardium glucose uptake, mitochondrial morphology disturbance, reduced activity of mitochondrial cytochrome c oxidase (COX) and lowered ATP content were detected after DOX stimulation in mice, which could be significantly improved by overexpression of COX5A. Overexpression of COX5A effectively protected against DOX-induced oxidative stress, mitochondrial dysfunction and cardiomyocyte apoptosis in vivo and in vitro. Mechanistically, the phosphorylation of Akt (Thr308) and Akt (Ser473) were also decreased following DOX treatment, which could be reserved by the upregulation of COX5A. Furthermore, PI3K inhibitors abrogated the protection effects of COX5A against DOX-induced cardiotoxicity in H9c2 cells. Thus, we identified that PI3K/Akt signaling was responsible for the COX5A-mediated protective role in DOX-induced cardiomyopathy. These results demonstrated the protective effect of COX5A in mitochondrial dysfunction, oxidative stress, and cardiomyocyte apoptosis, providing a potential therapeutic target in DOX-induced cardiomyopathy.

Translocase of outer mitochondrial membrane 40 (TOMM40) is located in the outer membrane of mitochondria. TOMM40 is essential for protein import into mitochondria. TOMM40 genetic variants are believed to increase the risk of Alzheimer’s disease (AD) in different populations. In this study, three exonic variants (rs772262361, rs157581, and rs11556505) and three intronic variants (rs157582, rs184017, and rs2075650) of the TOMM40 gene were identified from Taiwanese AD patients using next-generation sequencing. Associations between the three TOMM40 exonic variants and AD susceptibility were further evaluated in another AD cohort. Our results showed that rs157581 (c.339T > C, p.Phe113Leu, F113L) and rs11556505 (c.393C > T, p.Phe131Leu, F131L) were associated with an increased risk of AD. We further utilized cell models to examine the role of TOMM40 variation in mitochondrial dysfunction that causes microglial activation and neuroinflammation. When expressed in BV2 microglial cells, the AD-associated mutant (F113L) or (F131L) TOMM40 induced mitochondrial dysfunction and oxidative stress-induced activation of microglia and NLRP3 inflammasome. Pro-inflammatory TNF-α, IL-1β, and IL-6 released by mutant (F113L) or (F131L) TOMM40-activated BV2 microglial cells caused cell death of hippocampal neurons. Taiwanese AD patients carrying TOMM40 missense (F113L) or (F131L) variants displayed an increased plasma level of inflammatory cytokines IL-6, IL-18, IL-33, and COX-2. Our results provide evidence that TOMM40 exonic variants, including rs157581 (F113L) and rs11556505 (F131L), increase the AD risk of the Taiwanese population. Further studies suggest that AD-associated mutant (F113L) or (F131L) TOMM40 cause the neurotoxicity of hippocampal neurons by inducing the activation of microglia and NLRP3 inflammasome and the release of pro-inflammatory cytokines.

Lung cancer is one of the deadliest cancers worldwide, including in Taiwan. The poor prognosis of the advanced lung cancer lies in delayed diagnosis and non-druggable targets. It is worth paying more attention to these ongoing issues. Public databases and an in-house cohort were used for validation. The KM plotter was utilized to discover the clinical significance. GSEA and GSVA were adopted for a functional pathway survey. Molecular biological methods, including proliferation, migration, and the EMT methods, were used for verification. Based on public databases, the increased expression of Ladinin 1 (LAD1) was presented in tumor and metastatic sites. Furthermore, an in-house cohort revealed a higher intensity of LAD1 in tumor rather than in normal parts. The greater the expression of LAD1 was, the shorter the duration of lung adenocarcinoma (LUAD) patient survival. Moreover, the association of B3GNT3 with LAD1 affected the survival of LUAD patients. Functional analyses using GSEA and GSVA revealed the associations with survival, migration, invasion, and EMT. Biologic functions supported the roles of LAD1 in proliferation via the cell cycle and migration in EMT. This study reveals that LAD1 plays a major role in regulating proliferation and migration in lung cancer and impacts survival in LUAD. It is worth investing in further studies and in the development of drugs targeting LAD1.

Psoriasis is a chronic autoimmune disease, and until now, it remains an incurable disease. Therefore, the development of new drugs or agents that ameliorate the disease will have marketing potential. Taiwanofungus camphoratus (TC) is a specific fungus in Taiwan. It is demonstrated to have anticancer, anti-inflammation, and hepatoprotective effects. However, the effects of TC fermented extract on psoriasis are under investigation. In this research, we studied the ability of TC on antioxidative activity and the efficacy of TC on interleukin-17 (IL-17A)-induced intracellular oxidative stress, inflammation-relative, and proliferation-relative protein expression in human keratinocytes. The results of a DPPH radical scavenging assay, reducing power assay, and hydroxyl peroxide inhibition assay indicated that TC has a potent antioxidant ability. Furthermore, TC could reduce IL-17A-induced intracellular ROS generation and restore the NADPH level. In the investigation of pathogenesis, we discovered TC could regulate inflammatory and cell proliferation pathways via p-IKKα/p-p65 and p-mTOR/p-p70S6k signaling pathways in human keratinocytes. In conclusion, TC showed characteristics such as antioxidant, anti-inflammatory, and anti-psoriatic-associated responses. It is expected to be developed as a candidate for oxidative-stress-induced skin disorders or psoriasis treatment.

Hericium erinaceus (HE) is a common edible mushroom consumed in several Asian countries and considered to be a medicinal mushroom with neuroprotective effects. Erinacine A (EA) is a bioactive compound in Hericium erinaceus mycelium (HEM) that has been shown to have a neuroprotective effect against neurodegenerative diseases, e.g., Parkinson’s disease (PD). Although the etiology of PD is still unclear, neuroinflammation may play an important role in causing dopaminergic neuron loss, which is a pathological hallmark of PD. However, glial cell activation has a close relationship with neuroinflammation. Thus, this study aimed to investigate the anti-neuroinflammatory and neuroprotective effects of EA on lipopolysaccharide (LPS)-induced glial cell activation and neural damage in vitro and in vivo. For the in vitro experiments, glial cells, BV-2 microglial cells and CTX TNA2 astrocytes were pretreated with EA and then stimulated with LPS and/or IFN-γ. The expression of proinflammatory factors in the cells and culture medium was analyzed. In addition, differentiated neuro-2a (N2a) cells were pretreated with EA or HEM and then stimulated with LPS-treated BV-2 conditioned medium (CM). The cell viability and the amount of tyrosine hydroxylase (TH) and mitogen-activated protein kinases (MAPKs) were analyzed. In vivo, rats were given EA or HEM by oral gavage prior to injection of LPS into the substantia nigra (SN). Motor coordination of the rats and the expression of proinflammatory mediators in the midbrain were analyzed. EA pretreatment prevented LPS-induced iNOS expression and NO production in BV-2 cells and TNF-α expression in CTX TNA2 cells. In addition, both EA and HEM pretreatment significantly increased cell viability and TH expression and suppressed the phosphorylation of JNK and NF- κB in differentiated N2a cells treated with CM. In vivo, both EA and HEM significantly improved motor dysfunction in the rotarod test and the amphetamine-induced rotation test and reduced the expression of TNF-α, IL-1β and iNOS in the midbrain of rats intranigrally injected with LPS. The results demonstrate that EA ameliorates LPS-induced neuroinflammation and has neuroprotective properties.

Type 1 diabetes (T1D) is caused by the destruction of β cells in pancreatic islets by autoimmune T cells. Islet transplantation has been established as an effective treatment for T1D. However, the survival of islet grafts is often disrupted by recurrent autoimmunity. Alpha-lipoic acid (ALA) has been reported to have immunomodulatory effects and, therefore, may have therapeutic potential in the treatment of T1D. In this study, we investigated the therapeutic potential of ALA in autoimmunity inhibition. We treated non-obese diabetic (NOD) mice with spontaneous diabetes and islet-transplantation mice with ALA. The onset of diabetes was decreased and survival of the islet grafts was extended. The populations of Th1 cells decreased, and regulatory T cells (Tregs) increased in ALA-treated mice. The in vitro Treg differentiation was significantly increased by treatment with ALA. The adoptive transfer of ALA-differentiated Tregs into NOD recipients improved the outcome of the islet grafts. Our results showed that in vivo ALA treatment suppressed spontaneous diabetes and autoimmune recurrence in NOD mice by inhibiting the Th1 immune response and inducing the differentiation of Tregs. Our study also demonstrated the therapeutic potential of ALA in Treg-based cell therapies and islet transplantation used in the treatment of T1D.

Head and neck squamous cell carcinomas (HNSCCs) are the most common cancers of the head and neck, and their prevalence is rapidly increasing. HNSCCs present a clinical challenge because of their high recurrence rate, therapeutic resistance to radiation and chemotherapy drugs, and adverse effects. Hence, traditional Chinese herbal treatment may be advantageous to therapeutic strategies for HNSCCs. Danshen (Salvia miltiorrhiza), a well-known Chinese herb, has been extensively applied in treatments for various diseases, including cancer, because of its high degree of safety and low rate of adverse effects despite its unclear mechanism. Thus, we aimed to explore the possible anticancer effects and mechanisms of dihydroisotanshinone I (DT), a compound in danshen (extract from danshen), on HNSCCs. Three HNSCCs cell lines were used for in vitro studies, and a Detroit 562 xenograft mouse model was chosen for in vivo studies. Our in vitro results showed that DT could initiate apoptosis, resulting in cell death, and the p38 signaling partially regulated DT-initiated cell apoptosis in the Detroit 562 model. In the xenograft mouse model, DT reduced tumor size with no obvious adverse effect of hepatotoxicity. The present study suggests that DT is a promising novel candidate for anti-HNSCCs therapy.

Chikungunya virus (CHIKV) is a mosquito-transmitted infectious agent that causes an endemic or epidemic outbreak(s) of Chikungunya fever that is reported in almost all countries. This virus is an intense global threat, due to its high rate of contagion and the lack of effective remedies. In this study, we developed two baculovirus expression vector system (BEVS)-based approaches for the screening of anti-CHIKV drugs in Spodoptera frugiperda insect (Sf21) cells and U-2OS cells. First, structural protein of CHIKV was co-expressed through BEVS and thereby induced cell fusion in Sf21 cells. We used an internal ribosome entry site (IRES) to co-express the green fluorescent protein (EGFP) for identifying these fusion events. The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form syncytia. We identified that ursolic acid has potential anti-CHIKV activity in vitro, by using this approach. Second, BacMam virus-based gene delivery has been successfully applied for the transient expression of non-structural proteins with a subgenomic promoter-EGFP (SP-EGFP) cassette in U-2OS cells to act as an in vitro CHIKV replicon system. Our BacMam-based screening system has identified that the potential effects of baicalin and baicalein phytocompounds can inhibit the replicon activity of CHIKV in U-2OS cells. In conclusion, our results suggested that BEVS can be a potential tool for screening drugs against CHIKV.

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

Cisplatin, a widely used chemotherapy for the treatment of various tumors, is clinically limited due to its extensive nephrotoxicity. Inflammatory response in tubular cells is a driving force for cisplatin-induced nephrotoxicity. The plant-derived agents are widely used to relieve cisplatin-induced renal dysfunction in preclinical studies. Polysulfide and hydrogen sulfide (H2S) are ubiquitously expressed in garlic, and both of them are documented as potential agents for preventing and treating inflammatory disorders. This study was designed to determine whether polysulfide and H2S could attenuate cisplatin nephrotoxicity through suppression of inflammatory factors. In renal proximal tubular cells, we found that sodium tetrasulfide (Na2S4), a polysulfide donor, and sodium hydrosulfide (NaHS) and GYY4137, two H2S donors, ameliorated cisplatin-caused renal toxicity through suppression of the massive production of inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2). Mechanistically, the anti-inflammatory actions of Na2S4 and H2S may be mediated by persulfidation of signal transducer and activator of transcription 3 (STAT3) and inhibitor kappa B kinase β (IKKβ), followed by decreased phosphorylation of STAT3 and IKKβ. Moreover, the nuclear translocation of nuclear transcription factor kappa B (NF-κB), and phosphorylation and degradation of nuclear factor kappa B inhibitor protein alpha (IκBα) induced by cisplatin, were also mitigated by both polysulfide and H2S. In mice, after treatment with polysulfide and H2S donors, cisplatin-associated renal dysfunction was strikingly ameliorated, as evidenced by measurement of serum blood urea nitrogen (BUN) and creatinine levels, renal morphology, and the expression of renal inflammatory factors. Our present work suggests that polysulfide and H2S could afford protection against cisplatin nephrotoxicity, possibly via persulfidating STAT3 and IKKβ and inhibiting NF-κB-mediated inflammatory cascade. Our results might shed light on the potential benefits of garlic-derived polysulfide and H2S in chemotherapy-induced renal damage.

The global depression population is showing a significant increase. Hemerocallis fulva L. is a common Traditional Chinese Medicine (TCM). Its flower buds are known to have ability to clear away heat and dampness, detoxify, and relieve depression. Ancient TCM literature shows that its roots have a beneficial effect in calming the spirit and even the temper in order to reduce the feeling of melancholy. Therefore, it is inferred that the root of Hemerocallis fulva L. can be used as a therapeutic medicine for depression. This study aims to uncover the pharmacological mechanism of the antidepressant effect of Hemerocallis Radix (HR) through network pharmacology method. During the analysis, 11 active components were obtained and screened using ADME—absorption, distribution, metabolism, and excretion— method. Furthermore, 267 HR targets and 740 depressive disorder (DD) targets were gathered from various databases. Then protein−protein interaction (PPI) network of HR and DD targets were constructed and cluster analysis was applied to further explore the connection between the targets. In addition, gene ontology (GO) enrichment and pathway analysis was applied to further verify that the biological process related to the target protein is associated with the occurrence of depression disorder. In conclusion, the most important bioactive components—anthraquinone, kaempferol, and vanillic acid—can alleviate depression symptoms by regulating MAOA, MAOB, and ESR1. The proposed network pharmacology strategy provides an integrating method to explore the therapeutic mechanism of multi-component drugs on a systematic level.

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.

Overexpression of the amyloid precursor protein (APP) and the hyperphosphorylation of the tau protein are vital in the understanding of the cause of Alzheimer’s disease (AD). As a consequence, regulation of the expression of both APP and tau proteins is one important approach in combating AD. The APP and tau proteins can be targeted at the levels of transcription, translation and protein structural integrity. This paper reports the utilization of a bi-cistronic vector containing either APP or tau internal ribosome entry site (IRES) elements flanked by β-galactosidase gene (cap-dependent) and secreted alkaline phosphatase (SEAP) (cap-independent) to discern the mechanism of action of memantine, an N-methyl-d-aspartate (NMDA) receptor antagonist. Results indicate that memantine could reduce the activity of both the APP and tau IRES at a concentration of ~10 μM (monitored by SEAP activity) without interfering with the cap-dependent translation as monitored by the β-galactosidase assay. Western blot analysis of the tau protein in neuroblastoma (N2A) and rat hippocampal cells confirmed the halting of the expression of the tau proteins. We also employed this approach to identify a preparation named NB34, extracts of Boussingaultia baselloides (madeira-vine) fermented with Lactobacillus spp., which can function similarly to memantine in both IRES of APP and Tau. The water maze test demonstrated that NB34 could improve the spatial memory of a high fat diet induced neurodegeneration in apolipoprotein E-knockout (ApoE−/−) mice. These results revealed that the bi-cistronic vector provided a simple, and effective platform in screening and establishing the mechanistic action of potential compounds for the treatment and management of AD.

The objective of this study was to analyze the expression levels of IL-8 in serum and liver tissues from patients with chronic hepatitis B (CHB) infection and to investigate whether IL-8 may antagonize interferon-alpha (IFN-α) antiviral activity against HBV. IL-8 expression in serum was determined by enzyme linked immunosorbent assay (ELISA), and fluorescence-based quantitative real-time PCR (RT-qPCR) was used to measure IL-8 mRNA in peripheral blood mononuclear cells (PBMCs) in patients with CHB. IL-8 protein expression was detected in liver biopsy tissues by immunohistochemistry. In addition, the differences in serum IL-8 and PBMCs mRNA levels were also observed in patients with different anti-viral responses to IFN-α. Compared to normal controls, serum IL-8 protein and mRNA levels were increased in CHB patients, IL-8 levels were positively correlated with the severity of liver inflammation/fibrosis. Moreover, serum IL-8 protein and mRNA levels were positively correlated with serum alanine aminotransferase (ALT) level and negatively correlated with serum prealbumin (PA) level. IL-8 expression was mainly located in portal area of liver tissues and was increased with the severity of liver inflammation and fibrosis stage. The expression serum and mRNA levels of IL-8 in the CHB patients with a complete response to IFN-α are significantly lower than that of the patients with non-response to IFN-α treatment. It is suggested that IL-8 might play important roles in the pathogenesis of CHB. Moreover, interferon resistance may be related to the up-regulation of IL-8 expression in the patients did not respond to IFN-α treatment.

Vascular calcification can be enhanced by hyperglycemia. Elastin loss in tunica media promotes the osteogenic transformation of smooth muscle cells (SMCs) and involves arterial medial calcification (AMC) that is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes. Here, we tested whether hydrogen sulfide (H2S), an endogenous gaseous mediator, can prevent elastin loss and attenuate calcification induced by high glucose in SMCs. Calcification was induced by high glucose (4500 mg/L) in human aortic SMCs (HASMCs) under the condition of calcifying medium containing 10 mM β-glycerophosphate (β-GP). The experiments showed that NaHS (an H2S donor, 100 μM) mitigated the calcification of HASMCs treated with high glucose by decreasing calcium and phosphorus levels, calcium deposition and ALP activity and inhibited osteogenic transformation by increasing SMα-actin and SM22α, two phenotypic markers of smooth muscle cells, and decreasing core binding factor α-1 (Cbfα-1), a key factor in bone formation, protein expressions in HASMCs. Moreover, NaHS administration inhibited the activation of Stat3, cathepsin S (CAS) activity and its expression, but increased the level of elastin protein. Pharmacological inhibition or gene silencing Stat3 not only reversed elastin loss, but also attenuated CAS expression. Inhibition of CAS alleviated, while CAS overexpression exacerbated, elastin loss. Interestingly, overexpression of wild type (WT)-Stat3, but not its mutant C259S, elevated CAS protein expression and reduced elastin level. Moreover, NaHS induced S-sulfhydration in WT, but not in the C259S Stat3. These data suggest that H2S may directly regulate Cys259 residue in Stat3 and then impair its signaling function. Our data indicate that H2S may attenuate vascular calcification by upregulating elastin level through the inhibition of Stat3/CAS signaling.

Bacillus subtilis ATCC (American type culture collection) 6633 was found to biotransform ganoderic acid A (GAA), which is a major lanostane triterpenoid from the medicinal fungus Ganoderma lucidum. Five glycosyltransferase family 1 (GT1) genes of this bacterium, including two uridine diphosphate-dependent glycosyltransferase (UGT) genes, BsUGT398 and BsUGT489, were cloned and overexpressed in Escherichia coli. Ultra-performance liquid chromatography confirmed the two purified UGT proteins biotransform ganoderic acid A into a metabolite, while the other three purified GT1 proteins cannot biotransform GAA. The optimal enzyme activities of BsUGT398 and BsUGT489 were at pH 8.0 with 10 mM of magnesium or calcium ion. In addition, no candidates showed biotransformation activity toward antcin K, which is a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea. One biotransformed metabolite from each BsUGT enzyme was then isolated with preparative high-performance liquid chromatography. The isolated metabolite from each BsUGT was identified as ganoderic acid A-15-O-β-glucoside by mass and nuclear magnetic resonance spectroscopy. The two BsUGTs in the present study are the first identified enzymes that catalyze the 15-O-glycosylation of triterpenoids.

NVP-BEZ235 or BEZ235 is a dual inhibitor of adenosine triphosphate (ATP)-competitive phosphoinositide 3-kinase (PI3K)/mammalian-target-of-rapamycin (mTOR) and is promising for cancer treatment. Because it targets more than one downstream effector, a dual approach is promising for cancer treatment. The aim of this study was to evaluate the efficacy of NVP-BEZ235 in treating oral cavity squamous cell carcinoma (OSCC). Two human OSCC cell lines, SCC-4 and SCC-25, were used in this study. PI3K-AKT signaling, proliferation, and cell migratory and invasion capabilities of OSCC cells were examined. In NVP-BEZ235-treated SCC-4 and SCC-25 cells, the phosphorylation of 70-kDa ribosomal S6 kinase (p70S6K), but not mTOR, decreased within 24 h. NVP-BEZ235 inhibited OSCC-cell proliferation, migration, and invasion possibly by directly deregulating the phosphorylation of p70S6K. The phospho-p70S6K inhibitor mimicked the effects of NVP-BEZ235 for preventing proliferation and weakening the migratory and invasion abilities of SCC-4 and SCC-25 cells. This study further confirmed the effect of NVP-BEZ235 on OSCC cells and provided a new strategy for controlling the proliferation, migration, and invasion of OSCC cells using the phopho-p70S6K inhibitor.

Melanin is synthesized through a series of interactions catalyzed by melanogenic enzymes such as tyrosinase, dopachrome tautomerase (tyrosinase-related protein-2; TRP-2), and tyrosinase-related protein-1 (TRP-1). Tyrosinase plays a key role in catalysing the initial and limiting steps of melanogenesis. The melanin that results from melanogenesis has the protective effect of absorbing ultraviolet radiation. However, overproduction of melanin, in addition to altering the appearance of skin, may lead to skin disorders such as melasma, solar lentigo, and postinflammatory hyperpigmentation. Previous studies have revealed that sesamol is a strong antioxidant and a free radical scavenger. In this study, we investigated the effects of sesamol on the regulation of melanogenesis and related mechanisms in B16F10 cells. The results indicated that sesamol inhibited tyrosinase activity and melanogenesis induced by α-melanocyte-stimulating hormone (α-MSH) in B16F10 melanoma cells. Sesamol decreased the protein level of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, and TRP-1 by downregulating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathways that had been activated by α-MSH. Sesamol increased glycogen synthase kinase 3 beta (GSK3β), protein kinase B (AKT), and extracellular signal-related kinase (ERK) phosphorylation, thus inhibiting the transcription of MITF. Sesamol also inhibited melanin synthesis and tyrosinase expression by modulating ERK, phosphoinositide 3-kinase (PI3K)/AKT, p38, and c-Jun amino-terminal kinase (JNK) signalling pathways. These results indicate that sesamol acted as a potent depigmenting agent.

Chronic ultraviolet (UV) exposure may cause skin damage, disrupt skin barrier function, and promote wrinkle formation. UV induces oxidative stress and inflammation, which results in extracellular matrix degradation in the dermis and epidermal hyperplasia. Our previous study demonstrated that fisetin exerts photoprotective activity by inhibiting mitogen-activated protein kinase/activator protein-1/matrix metalloproteinases (MMPs) activation. In this study, fisetin was applied topically to investigate its antiphotodamage effects in hairless mice. The erythema index (a* values) and transepidermal water loss were evaluated to assess skin damage, and immunohistochemical staining was conducted to elucidate the photoprotective mechanism of fisetin. The results revealed that the topical application of fisetin reduced UVB-induced increase in the a* value and wrinkle formation. In addition, fisetin inhibited epidermal hyperplasia and increased the collagen content in the dermis. Fisetin exerted photoprotective activity by inhibiting the expression of MMP-1, MMP-2, and cyclooxygenase-2 and increasing the expression of nuclear factor erythroid 2-related factor. Furthermore, fisetin increased the expression of filaggrin to prevent UVB-induced barrier function disruption. Altogether, the present results provide evidence of the effects and mechanisms of fisetin’s antiphotodamage and antiphotoinflammation activities.

Coffea arabica extract (CAE) containing 48.3 ± 0.4 mg/g of chlorogenic acid and a trace amount of caffeic acid was found to alleviate photoaging activity in human skin fibroblasts. In this study, polyphenol-rich CAE was investigated for its antioxidant and antiinflammatory properties, as well as for its capability to alleviate ultraviolet B (UVB)-induced photodamage in BALB/c hairless mice. The results indicated that 500 μg/mL of CAE exhibited a reducing power of 94.7%, ferrous ion chelating activity of 46.4%, and hydroxyl radical scavenging activity of 20.3%. The CAE dose dependently reduced UVB-induced reactive oxygen species (ROS) generation in fibroblasts. Furthermore, CAE inhibited the UVB-induced expression of cyclooxygenase-2 and p-inhibitor κB, and the translocation of nuclear factor-kappa B (NF-κB) to the nucleus of fibroblasts. In addition, CAE alleviated UVB-induced photoaging and photodamage in BALB/c hairless mice by restoring the collagen content and reduced UVB-induced epidermal hyperplasia. CAE also inhibited UVB-induced NF-κB, interleukin-6, and matrix metalloproteinase-1 expression in the hairless mouse skin. The results indicated that CAE exhibits antiphotodamage activity by inhibiting UV-induced oxidative stress and inflammation. Therefore, CAE is a candidate for use in antioxidant, antiinflammatory, and antiphotodamage products.

BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy.

Here, dual-sensitive fluorescent branched alginate-polyethyleneimine copolymer (bAPSC) nanogels were synthesized from thiolated alginate and stearoyl-derivatized branched polyethyleneimine. The formation of bAPSC conjugates was confirmed through proton nuclear magnetic resonance and Fourier transform infrared spectroscopy, whereas dynamic light scattering was used to measure the particle size and ζ potential of the nanogels. The fluorescent properties of the nanogels were confirmed through fluorescent spectroscopy and microscopy. In addition to the excitation-dependent fluorescence behavior, the fluorescence emission intensity of bAPSC was altered by both pH and γ-irradiation. This intensity was higher at a lower pH than at a higher pH, and it slightly decreased after γ-irradiation. The drug loading and encapsulation efficiency of bAPSC were 25.9% and 11.2%, respectively. An in vitro drug release study revealed that the synthesized nanogels release their doxorubicin (Dox) contents in a time-dependent manner, and the drug release was higher after 96 h of incubation. Approximately 43.74% and 88.36% of Dox was released after 96 h of incubation at pH 5.5 in the absence and presence of glutathione (GSH), respectively. However, relatively lower drug release, approximately 21.6% and 16%, was observed in the presence and absence of GSH at pH 7.4, respectively. Fluorescence microscopy confirmed that Dox-loaded bAPSC nanogels were internalized by HeLa cells, and drug distribution was easily tracked using fluorescent materials without additional probing agents. Moreover, cellular cytotoxicity and hemolysis results revealed less cytotoxicity and hemocompatibility of the synthesized nanogels, confirming that they are the most favorable alternative drug carriers for drug delivery systems. [ABSTRACT FROM AUTHOR]