Silver Nanoparticles Encapped by Dihydromyricetin: Optimization of Green Synthesis, Characterization, Toxicity, and Anti-MRSA Infection Activities for Zebrafish (Danio rerio).

To achieve the environmentally friendly and rapid green synthesis of efficient and stable AgNPs for drug-resistant bacterial infection, this study optimized the green synthesis process of silver nanoparticles (AgNPs) using Dihydromyricetin (DMY). Then, we assessed the impact of AgNPs on zebrafish embryo development, as well as their therapeutic efficacy on zebrafish infected with Methicillin-resistant Staphylococcus aureus (MRSA). Transmission electron microscopy (TEM) and dynamic light-scattering (DLS) analyses revealed that AgNPs possessed an average size of 23.6 nm, a polymer dispersity index (PDI) of 0.197 ± 0.0196, and a zeta potential of -18.1 ± 1.18 mV. Compared to other published green synthesis products, the optimized DMY-AgNPs exhibited smaller sizes, narrower size distributions, and enhanced stability. Furthermore, the minimum concentration of DMY-AgNPs required to affect zebrafish hatching and survival was determined to be 25.0 µg/mL, indicating the low toxicity of DMY-AgNPs. Following a 5-day feeding regimen with DMY-AgNP-containing food, significant improvements were observed in the recovery of the gills, intestines, and livers in MRSA-infected zebrafish. These results suggested that optimized DMY-AgNPs hold promise for application in aquacultures and offer potential for further clinical use against drug-resistant bacteria.

The anti-inflammatory effect of ent-kaur-15-en-17-al-18-oic acid on lipopolysaccharide-stimulated RAW264.7 cells associated with NF-κB and P38/MAPK pathways.

Ent-kaur-15-en-17-al-18-oic acid (LL-3) was demonstrated that it can inhibit LPS-induced nitric oxide (NO) production and macrophage migration, maintain homeostasis of oxidative stress, including increased mitochondrial membrane potential (MMP), decreased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and maintenance of superoxide dismutase (SOD) and glutathione (GSH) activities and inhibit oxidative stress-induced P38 and nuclear factor κB (NF-κB) pathways to decrease inducible nitric oxide synthase (iNOS), cyclooxygense-2 (COX-2), and tumour necrosis factor (TNF)-α mRNA expressions without marked cytotoxicity. These findings revealed that LL-3 could serve as a candidate lead compound for further studying anti-inflammatory therapies.[Formula: see text].

Involvement of Reactive Oxygen Species in the Hepatorenal Toxicity of Actinomycin V In Vitro and In Vivo.

The high toxicity of actinomycin D (Act D) severely limits its use as a first-line chemotherapeutic agent in the clinic. Actinomycin V (Act V), an analog of Act D, exhibited strong anticancer activity in our previous studies. Here, we provide evidence that Act V has less hepatorenal toxicity than Act D in vitro and in vivo, associated with the reactive oxygen species (ROS) pathway. Compared to Act D, Act V exhibited considerably stronger sensitivity for cancer cells and less toxicity to human normal liver LO-2 and human embryonic kidney 293T cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. Notably, Act V caused less damage to both the liver and kidney than Act D in vivo, indicated by organ to body weight ratios, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (Scr) levels. Further experiments showed that the ROS pathway is involved in Act V-induced hepatorenal toxicity. Act V generates ROS and accumulates malondialdehyde (MDA), reducing levels of superoxide dismutase (SOD) and glutathione (GSH) in LO-2 and 293T cells. These findings indicate that Act V induces less hepatorenal toxicity than Act D in vitro and in vivo and merits further development as a potential therapeutic agent for the treatment of cancer.

Hexokinase II Inhibitory Effect of Secondary Metabolites Derived from a Streptomyces sp. Associated with Mud Dauber Wasp.

Insect-microbial symbioses have vast biochemical diversity, which is beneficial to produce bioactive secondary metabolites. In this study, chemical examination of a Streptomyces sp. associated with a mud dauber wasp led to the isolation of fourteen compounds. Their structures were determined by spectroscopic methods and comparison with literature data. Among the isolates, compounds 1,2,3-benzotriazin-4(1H)-one and 4-(2-aminoethyl)phenyl acetate were first reported from this species. Bioactivities of the isolated compounds were assayed for the first time against hexokinase II. 4-(2-Aminoethyl)phenyl acetate, germicidin B, phenylacetic acid, isogermicidin A and germicidin C displayed significant inhibitory activity against hexokinase II, with the IC50 values of 5.11, 7.11, 7.15, 8.45 and 8.78 µM, respectively.

Strepantibins A-C: Hexokinase II Inhibitors from a Mud Dauber Wasp Associated Streptomyces sp.

Two new p-terphenyls, strepantibins A and B (1 and 2), along with the first representative of a naturally occurring bisphenyltropone, strepantibin C (3), were characterized from a Streptomyces sp. associated with the larvae of the mud dauber wasp Sceliphron madraspatanum. Their structures were determined by high-resolution electrospray ionization mass spectrometry, NMR, and X-ray crystallography data interpretation. Strepantibins A-C inhibited hexokinase II (HK2) activity and displayed antiproliferative activity against hepatoma carcinoma cells HepG-2, SMMC-7721 and plc-prf-5. In SMMC-7721 cells treated with strepantibin A, the morphological characteristics of apoptosis were observed.

Actinomycin V Suppresses Human Non-Small-Cell Lung Carcinoma A549 Cells by Inducing G2/M Phase Arrest and Apoptosis via the p53-Dependent Pathway.

Actinomycin V, extracted and separated from marine-derived actinomycete Streptomyces sp., as the superior potential replacement of actinomycin D (which showed defect for its hepatotoxicity) has revealed an ideal effect in the suppression of migration and invasion in human breast cancer cells as referred to in our previous study. In this study, the involvement of p53 in the cell cycle arrest and pro-apoptotic action of actinomycin V was investigated in human non-small-cell lung carcinoma A549 cells. Results from the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay showed that cytotoxic activity of actinomycin V on A549 cells (with wild-type p53) was stronger than the NCI-H1299 cells (p53-deficient). Actinomycin V upregulated both of the protein and mRNA expression levels of p53, p21Waf1/Cip1 and Bax in A549 cells. For this situation, actinomycin V decreased the M-phase related proteins (Cdc2, Cdc25A and Cyclin B1) expression, arrested cells in G2/M phase and subsequently triggered apoptosis by mediating the Bcl-2 family proteins' expression (Bax and Bcl-2). Furthermore, the effects of cell cycle arrest and apoptosis in A549 cells which were induced by actinomycin V could be reversed by the pifithrin-α, a specific inhibitor of p53 transcriptional activity. Collectively, our results suggest that actinomycin V causes up-regulation of p53 by which the growth of A549 cells is suppressed for cell cycle arrest and apoptosis.

Cytotoxic, Anti-Migration, and Anti-Invasion Activities on Breast Cancer Cells of Angucycline Glycosides Isolated from a Marine-Derived Streptomyces sp.

Four angucycline glycosides were previously characterized from marine-derived Streptomyces sp. OC1610.4. Further investigation of this strain cultured on different fermentation media from that used previously resulted in the isolation of two new angucycline glycosides, vineomycins E and F (1-2), and five known homologues, grincamycin L (3), vineomycinone B2 (4), fridamycin D (5), moromycin B (7), and saquayamycin B1 (8). Vineomycin F (2) contains an unusual ring-cleavage deoxy sugar. All the angucycline glycosides isolated from Streptomyces sp. OC1610.4 were evaluated for their cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231, and BT-474. Moromycin B (7), saquayamycin B1 (8), and saquayamycin B (9) displayed potent anti-proliferation against the tested cell lines, with IC50 values ranging from 0.16 to 0.67 µM. Saquayamycin B (9) inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner, as detected by Transwell and wound-healing assays.

Protective effects of 3ß-angeloyloxy-8ß, 10ß-dihydroxyeremophila-7(11)-en-12, 8α-lactone on paraquat-induced oxidative injury in SH-SY5Y cells.

3ß-Angeloyloxy-8ß,10ß-dihydroxyeremophila-7(11)-en-12,8α-lactone (FJ1) inhibited effectively paraquat (PQ)-induced injury in SH-SY5Y cells. In this way, FJ1 was shown to reverse the PQ-induced activation of caspase-9 and caspase-3, the increase in Bax/Bcl-2 ratio, and the release of cytochrome c. The mechanism was associated with a reduction of oxidative stress, including the decrease in the levels of ROS and MDA and maintaining the activity of SOD and GSH. Taken together, findings revealed that FJ1 had protective effects against PQ-induced injury via attenuating the oxidative stress in SH-SY5Y cells, which suggested that FJ1 might be a candidate for further evaluation against neurodegeneration in Parkinson's disease.

Ivalin Induces Mitochondria-Mediated Apoptosis Associated with the NF-κB Activation in Human Hepatocellular Carcinoma SMMC-7721 Cells.

Ivalin, a natural compound isolated from Carpesium divaricatum, showed excellent microtubule depolymerization activities among human hepatocellular carcinoma in our previous work. Here, we investigated its functions on mitochondria-mediated apoptosis in hepatocellular carcinoma SMMC-7721 cells. DAPI (4',6-diamidino-2-phenylindole) staining, annexin V-fluorexcein isothiocyanate (FITC) apoptosis detection, and western blotting were applied to explore the apoptotic effect of Ivalin. Next, the induction effect of Ivalin on the mitochondrial pathway was also confirmed via a series of phenomena including the damage of mitochondria membrane potential, mitochondria cytochrome c escape, cleaved caspase-3 induction, and the reactive oxygen species generation. In this connection, we understood that Ivalin induced apoptosis through the mitochondrial pathway and the overload of reactive oxygen species. Furthermore, we found that the activation of nuclear factor-κB (NF-κB) and subsequent p53 induction were associated with the apoptotic effect of Ivalin. These data confirmed that Ivalin might be a promising pro-apoptotic compound that can be utilized as a potential drug for clinical treatment.

Ivalin Inhibits Proliferation, Migration and Invasion by Suppressing Epithelial Mesenchymal Transition in Breast Cancer Cells.

Ivalin, an eudesmane-type sesquiterpene compound, was isolated from the Chinese herb Carpesium divaricatum in our chemistry group. In this study, we investigated the anti-migration and anti-invasion activities and underlying mechanisms of Ivalin in breast cancer cells in vitro. Cell viability was evaluated using the MTT assay, Western blotting was used to determine the expression of E-cadherin, N-cadherin, vimentin and ZEB1, and mRNA levels were analyzed by qPCR. The anti-migration and anti-invasion effects of Ivalin were measured by wound-healing and Transwell assays. In this connection, Ivalin treatment reduced the mRNA and protein expressions of ZEB1 as well as N-cadherin and vimentin expression in various breast cancer cells. E-cadherin expression was enhanced by Ivalin in the same cells, which implied that Ivalin depressed the process of epithelial-to-mesenchymal transition (EMT). Our results revealed that Ivalin significantly inhibited cell proliferation, migration and invasion in breast cancer cells in a dose-dependent manner in vitro. This study suggests that Ivalin may merit further investigation as a potential therapeutic leading compound for the treatment of breast cancer migration and invasion.

5,6-Dihydroxy-3,7,4'-trimethoxyflavonol induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma cells.

5,6-Dihydroxy-3,7,4'-trimethoxyflavonol (AH5), 5,6,3'-trihydroxy-3,7,4'-trimethoxyflavonol (AH22), artemetin, and oroxylin A are four flavonoids with the same 2-phenyl-chromone skeleton isolated from the Chinese herb Aster himalaicus. The aim of this study was to evaluate the structure-activity relationship of these four analogs and the mediation of AH5 cytotoxicity via G2/M arrest and apoptosis in human hepatocellular carcinoma (HCC) cells. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated AH5 showed the better potency to inhibit proliferation in human HCC cells, which suggested hydroxyl binding to C6 is necessary to anticancer properties, whereas binding to C3' attenuated the activities and increased toxicity in tested cells. Flow cytometry analysis revealed that AH5-induced G2/M arrest and significantly apoptosis in these cell lines. HepG-2 cells were used to further evaluate the antitumor effects and mechanisms of AH5. AH5-induced apoptosis was further confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining and the increased ratio of Bax/Bcl-2. Moreover, AH5 induced the release of cytochrome C and the activation of caspase-9 and caspase-3, thus suggesting mitochondria activation might be involved. Western blot showed that AH5 induced the phosphorylation of Cdc2 and decreased the level of Cyclin B1. These results demonstrated that AH5 could be a proapoptotic leading compound for developing novel anticancer drugs.

Telekin suppresses human hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via the p38 MAPK signaling pathway.

AIM: Telekin, isolated from the Chinese herb Carpesium divaricatum, has shown anti-proliferation effects against various cancer cells, including hepatocellular carcinoma cells. In this study, we investigated the anti-proliferation mechanisms of telekin in human hepatocellular carcinoma HepG2 cells in vitro. METHODS: HepG2 cells were treated with telekin. Cell viability was evaluated using MTT assay. Flow cytometry was used to measure cell cycle profiles, ROS level and apoptosis. The protein expression levels were analyzed with Western blotting. RESULTS: Telekin (3.75-30 µmol/L) dose-dependently inhibited the viability of HepG2 cells and induced l apoptosis. Furthermore, the treatment induced cell cycle arrest at G2/M phase, accompanied by significantly increased the phosphorylation of Cdc25A and Cdc2, and decreased Cyclin B1 level. Moreover, the treatment significantly stimulated ROS production, and increased the phosphorylation of p38 and MAPKAPK-2 in the cells. Pretreatment with the antioxidant NAC (2.5, 5, and 10 mmol/L), or the p38 MAPK inhibitor SB203580 (2.5 and 5 µmol/L) dose-dependently attenuated these telekin-induced effects in the cells. CONCLUSION: Telekin suppresses hepatocellular carcinoma cells in vitro by inducing G2/M phase arrest via activating the p38 MAPK pathway.

1-Oxoeudesm-11(13)-eno-12,8a-lactone induces G2/M arrest and apoptosis of human glioblastoma cells in vitro.

AIM: To investigate the effects of 1-oxoeudesm-11(13)eno-12,8a-lactone (OEL), a novel eudesmane-type sesquiterpene isolated from Aster himalaicus, on the cell cycle and apoptosis in human glioblastoma cells in vitro. METHODS: Human malignant glioblastoma cell lines U87 and A172 were used. The cytotoxicity of OEL was examined using the MTT assay. Cell apoptosis was assessed with DAPI staining and flow cytometry. DNA damage was determined by measuring the phosphorylation of H2AX using immunofluorescence staining and Western blotting. Cell cycle profiles were measured with flow cytometry. The mRNA expression of p53 and p21Waf1/Cip1 was investigated using real-time PCR. The protein expression of γ-H2AX, caspase-9, caspase-3, p53, p21Waf1/Cip1, cyclin B1, and cdc2 was analyzed with Western blotting. RESULTS: Treatment of the malignant glioblastoma cells with OEL inhibited the cell growth in dose- and time-dependent manners (the values of IC(50) at 48 and 72 h were 29.5 and 16.99 µmol/L, respectively, in U87 cells; 7.2 and 9.5 µmol/L, respectively, in A172 cells). OEL (10-30 µmol/L) induced apoptosis and G(2)/M phase arrest in both U87 and A172 cells. OEL induced the phosphorylation of cdc2, a G(2)/M phase cyclin-dependent kinase, and decreased the expression of cyclin B1 required for progression through the G(2)/M phase in U87 cells. The compound remarkably increased the phosphorylation of H2AX in U87 cells. Moreover, OEL increased the mRNA and protein levels of p53 and its target gene p21(Waf1/Cip1) in U87 cells. The compound also induced p53 phosphorylation. Pretreatment with PFT-α, a specific inhibitor of p53 transcriptional activity, could partially reverse the inhibition of OEL on the viability of U87 and A172 cells. CONCLUSION: OEL suppresses the growth of human glioblastoma cells in vitro via inducing DNA damage, p53-mediated cell cycle arrest and apoptosis, thus warrants further studies as a lead compound of anti-glioblastoma drug.

Altered GC- and AT-biased genotypes of Ophiocordyceps sinensis in the stromal fertile portions and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine multiple genotypes of Ophiocordyceps sinensis in a semi-quantitative manner in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis and to outline the dynamic alterations of the coexisting O. sinensis genotypes in different developmental phases. METHODS: Mature Cordyceps sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,254 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. RESULTS: Microscopic analysis revealed distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown upstream and downstream sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same Cordyceps sinensis specimens. CONCLUSION: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure, and the two types of ascospores of Cordyceps sinensis, demonstrating their genomic independence. Metagenomic fungal members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural Cordyceps sinensis.

Differential coexistence of multiple genotypes of Ophiocordyceps sinensis in the stromata, ascocarps and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine the differential occurrence of Ophiocordyceps sinensis genotypes in the stroma, stromal fertile portion (SFP) densely covered with numerous ascocarps, and ascospores of natural Cordyceps sinensis. METHODS: Immature and mature C. sinensis specimens were harvested. Mature C. sinensis specimens were continuously cultivated in our laboratory (altitude 2,200 m). The SFPs (with ascocarps) and ascospores of C. sinensis were collected for microscopic and molecular analyses using species-/genotype-specific primers. Sequences of mutant genotypes of O. sinensis were aligned with that of Genotype #1 Hirsutella sinensis and compared phylogenetically using a Bayesian majority-rule method. RESULTS: Fully and semiejected ascospores were collected from the same specimens. The semiejected ascospores tightly adhered to the surface of the asci as observed by the naked eye and under optical and confocal microscopies. The multicellular heterokaryotic ascospores showed uneven staining of nuclei. The immature and mature stromata, SFPs (with ascocarps) and ascospores were found to differentially contain several GC- and AT-biased genotypes of O. sinensis, Samsoniella hepiali, and an AB067719-type fungus. The genotypes within AT-biased Cluster-A in the Bayesian tree occurred in all compartments of C. sinensis, but those within AT-biased Cluster-B were present in immature and mature stromata and SPFs but absent in the ascospores. Genotype #13 of O. sinensis was present in semi-ejected ascospores and Genotype #14 in fully ejected ascospores. GC-biased Genotypes #13-14 featured large DNA segment substitutions and genetic material recombination between the genomes of the parental fungi (H. sinensis and the AB067719-type fungus). These ascosporic offspring genotypes combined with varying abundances of S. hepiali in the 2 types of ascospores participated in the control of the development, maturation and ejection of the ascospores. CONCLUSION: Multiple genotypes of O. sinensis coexist differentially in the stromata, SFPs and 2 types of C. sinensis ascospores, along with S. hepiali and the AB067719-type fungus. The fungal components in different combinations and their dynamic alterations in the compartments of C. sinensis during maturation play symbiotic roles in the lifecycle of natural C. sinensis.

Candidate Circulating Biomarkers of Spontaneous Miscarriage After IVF-ET Identified via Coupling Machine Learning and Serum Lipidomics Profiling.

Spontaneous miscarriage is a common pregnancy complication. Multiple etiologies have been proposed such as genetic aberrations, endocrinology disorder, and immunologic derangement; however, the relevance of circulating lipidomes to the specific condition remains unclear. In the present study, lipidomics profiling was examined on serum of women with spontaneous miscarriage after in vitro fertilization and embryo transfer (IVF-ET). Screening and analysis of differential lipid levels were conducted using a machine learning approach to verify the stability and validity of potential serum biomarkers. Seven lipid species presented significant differences between the abortion and term birth patients, including three types of sphingomyelins (SMs), two types of diglycerides (DGs), one phosphatidylcholine (PC), and one lysophosphatidylethanolamine (LPE). All the SMs presented with a fold change of > 1, while both the PC and LPE had a fold change of < 1. The DG containing two saturated fatty acyl chains was decreased, but that containing two unsaturated fatty acyl chains was increased in the miscarriage group compared to the control group. This study reveals the relevance of lipid profiles to spontaneous abortion after IVF-ET, providing potential biomarkers and therapeutic targets for the specific clinical scenario.

MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is a complex process, which is regulated by various factors including microRNAs. Our preliminary data showed that the expression of endogenous miR-20a was increased during the course of osteogenic differentiation. Simultaneously, the expression of osteoblast markers and regulators BMP2, BMP4, Runx2, Osx, OCN and OPN was also elevated whereas adipocyte markers PPARγ and osteoblast antagonist, Bambi and Crim1, were downregulated, thereby suggesting that miR-20a plays an important role in regulating osteoblast differentiation. To validate this hypothesis, we tested its effects on osteogenic differentiation by introducing miR-20a mimics and lentiviral-miR20a-expression vectors into hMSCs. We showed that miR-20a promoted osteogenic differentiation by the upregulation of BMP/Runx2 signaling. We performed bioinformatics analysis and predicted that PPARγ, Bambi and Crim1 would be potential targets of miR-20a. PPARγ is a negative regulator of BMP/Runx2 signaling whereas Bambi or Crim1 are antagonists of the BMP pathway. Furthermore, we confirmed that all these molecules were indeed the targets of miR-20a by luciferase reporter, quantitative RT-PCR and western blot assays. Similarly to miR-20a overexpression, the osteogenesis was enhanced by the silence of PPARγ, Bambi or Crim1 by specific siRNAs. Taken together, for the first time, we demonstrated that miR-20a promoted the osteogenesis of hMSCs in a co-regulatory pattern by targeting PPARγ, Bambi and Crim1, the negative regulators of BMP signaling.

Fischerisin A and B, cytotoxic sesquiterpenoid-geranylhydroquinones from Ligularia fischeri.

During the course of screening natural sesquiterpenoids for new antitumor agents, two novel compounds, fischerisin A (1) and fischerisin B (2), were isolated from the roots of Ligularia fischeri. Their structures were elucidated by interpretation of their IR, high resolution-mass spectrometry (HR-MS), 1D- and 2D-NMR data. Fischerisin A and B are the first representatives of a novel sesquiterpenoid-geranylhydroquinone hybrid, and both compounds exhibited in vitro cytotoxicity against cultured human oral epidermoid carcinoma (KB) and human breast cancer (MCF-7) cell lines with IC(50) values of 9.7 and 10.2 µM, and 9.8 and 17.8 µM, respectively.