Sintilimab with two cycles of chemotherapy for the treatment of advanced squamous non-small cell lung cancer: a phase 2 clinical trial.

This was a single-arm, multicenter phase 2 clinical trial (ChiCTR1900021726) involving advanced squamous non-small cell lung cancer (sq-NSCLC) patients undergoing 2 cycles of nab-paclitaxel/carboplatin and sintilimab (anti-PD-1), followed by sintilimab maintenance therapy. The median progression-free survival (PFS) was 11.4 months (95% CI: 6.7-18.1), which met the pre-specified primary endpoint. Secondary endpoints included objective response rate reaching 70.5% and a disease control rate of 93.2%, with a median duration of response of 13.6 months [95% CI: 7.0-not evaluable (NE)]. The median overall survival was 27.2 months (95% CI: 20.2-NE) with treatment-related adverse events grades ≥3 occurring in 10.9% of patients. Predefined exploratory endpoints comprised relationships between biomarkers and treatment efficacy, and the association between circulating tumor DNA (ctDNA) dynamics and PFS. Biomarker analysis revealed that the breast cancer gene 2, BMP/Retinoic Acid Inducible Neural Specific 3, F-box/WD repeat-containing protein 7, tyrosine-protein kinase KIT and retinoblastoma 1 abnormalities led to shorter PFS, while ctDNA negative at baseline or clearance at 2 cycles of treatment was associated with longer PFS (18.1 vs. 4.3 months). Taken together, sintilimab in combination with 2 cycles of nab-paclitaxel/carboplatin treatment produced encouraging PFS and better tolerability as first-line treatment for advanced sq-NSCLC.

SARS-CoV-2 vaccine breakthrough infection in the older adults: a meta-analysis and systematic review.

BACKGROUND: Corona Virus Disease 2019 (COVID-19) mRNA vaccine effectiveness (VE) has recently declined, and reports about COVID-19 breakthrough infection have increased. We aimed to conduct a meta-analysis on population-based studies of the prevalence and incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) breakthrough infection amongst older adults worldwide. METHODS: Studies from PubMed, Embase, Cochrane Library, and Web of Science were systematically screened to determine the prevalence and incidence of SARS-CoV-2 breakthrough infection in older adults from inception to November 2, 2022. Our meta-analysis included 30 studies, all published in English. Pooled estimates were calculated using a random-effect model through the inverse variance method. Publication bias was tested through funnel plots and Egger's regression test, and sensitivity analyses were performed to confirm the robustness of the results. This research was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Thirty publications were included in this meta-analysis (17 on prevalence, 17 on incidence, and 4 on both). The pooled prevalence of COVID-19 breakthrough infection among older adults was 7.7 per 1,000 persons (95% confidence interval [95%CI] 4.0-15.0). At the same time, the pooled incidence was 29.1 per 1000 person-years (95%CI 15.2-55.7). CONCLUSIONS: This meta-analysis provides estimates of prevalence and incidence in older adults. We concluded that the prevalence and incidence of SARS-CoV-19 breakthrough infection in older people was low. The prevalence and incidence of breakthrough infection admitted to hospital, severe-critical, and deathly was significantly lower. Otherwise, there was considerable heterogeneity among estimates in this study, which should be considered when interpreting the results.

SERS-active plasmonic metal NP-CsPbX3 films for multiple veterinary drug residues detection.

Sensitive and multiple veterinary drug residues detection is of important for food safety. Herein, uniform plasmonic Au nanobipyramids@Ag nanorods (Au NBP@Ag NR)-CsPbX3 films with strong surface-enhanced Raman scattering (SERS) activity were constructed. The effects of different CsPbX3 (X = Cl, Br, I, or mixed halogens) quantum dots (QDs) on the SERS performances of plasmonic metal NP films were investigated. CsPbI3 QDs with large dielectric constant could be served as the dielectric media to retard the attenuation of electromagnetic evanescent wave, inducing strong electromagnetic strength for SERS enhancement. Plasmon-induced metal-to-perovskite interfacial charge transfer transition also contributed to SERS enhancement. SERS-active plasmonic Au NBP@Ag NR-CsPbI3 films had excellent sensitivity and high reproducibility for quantitative, accurate and multiple detection of chloramphenicol, diazepam and malachite green in food matrix. This work deepened the understanding of the SERS enhancement mechanisms of plasmonic metal NP-perovskite hybrid heterostructures, showing potential prospects in food safety monitoring.

A natural allele of OsMS1 responds to temperature changes and confers thermosensitive genic male sterility.

Changes in ambient temperature influence crop fertility and production. Understanding of how crops sense and respond to temperature is thus crucial for sustainable agriculture. The thermosensitive genic male-sterile (TGMS) lines are widely used for hybrid rice breeding and also provide a good system to investigate the mechanisms underlying temperature sensing and responses in crops. Here, we show that OsMS1 is a histone binding protein, and its natural allele OsMS1wenmin1 confers thermosensitive male sterility in rice. OsMS1 is primarily localized in nuclei, while OsMS1wenmin1 is localized in nuclei and cytoplasm. Temperature regulates the abundances of OsMS1 and OsMS1wenmin1 proteins. The high temperature causes more reduction of OsMS1wenmin1 than OsMS1 in nuclei. OsMS1 associates with the transcription factor TDR to regulate expression of downstream genes in a temperature-dependent manner. Thus, our findings uncover a thermosensitive mechanism that could be useful for hybrid crop breeding.

SERS-Active Composites with Au-Ag Janus Nanoparticles/Perovskite in Immunoassays for Staphylococcus aureus Enterotoxins.

The accurate detection of Staphylococcus aureus enterotoxins (SEs) is vital for food safety owing to their high pathogenicity, which may be performed with surface-enhanced Raman scattering (SERS) if SERS-active nanostructures are used. Herein, a Au-Ag Janus nanoparticle (NPs)/perovskite composite-engineered SERS immunoassay was developed for SEC detection. Plasmonic Au-Ag Janus NPs demonstrated inherent SERS activity from the 2-mercaptobenzoimidazole-5-carboxylic acid ligands. CsPbBr3@mesoporous silica nanomaterials (MSNs) were prepared and transformed into CsPb2Br5@MSNs in the aqueous phase. Paired SEC antibody-antigen-driven plasmonic Au-Ag Janus NP-CsPb2Br5@MSN composites were prepared. They showed amplified SERS activity, attributed to the depressed plasmonic decay due to electromagnetic field enhancement and the electron transfer mechanism. A positive relationship was established between SERS signals of composites and the SEC concentration. An additive-free SERS immunoassay was developed for simple, sensitive, and reproducible SEC detection. This study will be extended to develop multiple additive-free SERS-active plasmonic NP/perovskite composites that will open up the possibility of exploring more SERS detection probes for food safety monitoring.

Surface-Enhanced Raman Scattering-Active Plasmonic Metal Nanoparticle-Persistent Luminescence Material Composite Films for Multiple Illegal Dye Detection.

Uniform two-dimensional plasmonic nanoparticle (NP)-semiconductor composite films could retard the attenuation of electromagnetic evanescent wave and show intensive Raman activity for the multiplex monitoring of hazards in a practical food matrix. Here, an efficient Raman platform is developed by employing a plasmonic nanoparticle (NP)-persistent luminescence material (PLM) composite film. PLM show upconversion photoluminescence (UCPL) properties. The emitted photons are absorbed by plasmonic NPs, which further boost the surface plasmon resonance for the generation of high polarizability and induce strong electromagnetic strength for surface-enhanced Raman scattering (SERS) enhancement. A UCPL-assisted SERS-enhanced mechanism is proposed and verified. A plasmonic NP-PLM film with superior SERS activity and detection capability becomes an alternative candidate for the sensitive and multiple detection of illegal addition of dyes in a food matrix. The proposed UCPL-assisted SERS-enhanced mechanism provides promising future directions to this end to design a next-generation SERS-active plasmonic NP-PLM composite film for the specific detection in complex samples.

"Add on" Dual-Modal Optical Immunoassay by Plasmonic Metal NP-Semiconductor Composites.

Staphylococcus aureus enterotoxins (SEs, involving SEA, SEB, SEC, SED, and SEE) are considered to be the common toxins causing food poisoning and are not allowed to be detected in food. Accurate and anti-interfering SE detection in a complex food matrix is urgently required for food safety. Dual-modal optical sensors are able to avoid mutual interference of optical signals and possess the advantages of high accuracy and sensitivity. Herein, Au nanobipyramids (Au NBPs) and persistent luminescence ZnGeGaO:Cr,Er,Yb nanoparticle (ZGGO NP) nanocomposites are fabricated using the SEC antibody/antigen as templates, which display enhanced persistent luminescence (PL) and surface-enhanced Raman scattering (SERS) strength. The enhanced PL of Au NBP-ZGGO NP nanocomposites is ascribed to plasmon-enhanced radiative transitions. It is first found that ZGGO NPs display unique upconversion fluorescence, which can be absorbed by Au NBPs and that they largely excite the intensive electromagnetic field for SERS enhancement. Dual-model optical immunoassay achieved anti-interfering and specific SEC detection with a limit of detection of 7.5 pg/mL for the PL signal and 8.9 pg/mL for the SERS signal in the range of 10 pg/mL-100 ng/mL. Depending on the plasmon-enhanced PL mechanism and upconversion fluorescence-enhanced SERS principle, plasmonic NP-semiconductor composites show potential prospects in the establishment of multimodal optical biosensors for the quantitative and accurate evaluation of analytes in a complex food matrix.

The recombinant Newcastle disease virus Anhinga strain expressing human TRAIL exhibit antitumor effects on a glioma nude mice model.

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. However, the potential oncolytic ability of the recombinant newcastle disease virus (NDV) Anhinga strain carried with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has not been fully explored at present. In the present study, the recombinant NDV/Anh-TRAIL that secretes soluble TRAIL was constructed and the experiment results suggested NDV/Anh-TRAIL as a promising candidate for glioma therapy. Growth kinetic and TRAIL secreted quantity of recombinant NDV/Anh-TRAIL virus were measured. Cytotoxic and cell apoptosis were analyzed for its anti-glioma therapy in vitro. Nude mice were used for the in vivo evaluation. Both tumor volume and mice behavior after injection were observed. The recombinant virus replicated with the same kinetics as the parental virus and the highest expression of TRAIL (77.8 ng/L) was found at 48 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole and flow cytometry data revealed that the recombinant NDV/Anh-TRAIL (56.1 ± 8.2%) virus could induce a more severe apoptosis rate, when compared with the NDV wild type (37.2 ± 7.0%) and mock (7.0 ± 1.8%) groups (P < .01), in U251 cells. Furthermore, in the present animal study, the average tumor volume was smaller in the NDV/Anh-TRAIL group (97.21 mm3 ), when compared with the NDV wild type (205.03 mm3 , P < .05) and PBS (310.30 mm3 , P < .01) groups.

Synthesis and Characterization of Enhanced Photocatalytic Activity with Li+-Doping Nanosized TiO2 Catalyst.

The photocatalytic efficiency of TiO2 is reduced by rapid electron-hole recombination. An effective approach to address this limitation is to have TiO2 doped with various metal ions or heteroatoms. Herein, we prepared a series of Li+-doped TiO2 nanoparticles showing high photocatalytic activities through the sol-gel method. The samples were characterized by X-ray diffraction (XRD) and surface area analyses. Effects of Li+ doping on the Brunauer-Emmett-Teller (BET) surface area, crystallite size, phase transformation temperature, and phase composition were studied. The results showed that Li+ doping can promote the generation of the rutile crystal phase in TiO2, lower the anatase-to-rutile transformation temperature, and generate the mixed-crystal effect. The photocatalytic degradation of methyl orange (MO) was used as a probe reaction to evaluate the photoactivity of the nanoparticles. Parameters affecting the photocatalytic efficiency, including the Li+ doping amount, calcination temperature, and catalyst amount, as well as the kinetics of the photocatalytic process toward the degradation of MO, were investigated. The mixed-crystal TiO2, which was doped with 1.0 mol % Li+ and calcined at 550 °C containing 27.1% rutile and 72.9% anatase phase, showed a 2.2-fold increase in the photoactivity on the basis of the rate constant of MO decomposition as compared with the undoped TiO2. The existence of a definite quantity of rutile phase could effectively inhibit the recombination of the electron-hole pairs, thus promoting photocatalytic activity.

Ratiometric persistent luminescence aptasensors for carcinoembryonic antigen detection.

NIR-emitted ZnGa2O4:Cr3+ persistent luminescence nanoparticles (ZGC NPs) coated with polydopamine (ZGC@PDA NPs) were designed featuring internal reference and quenching ability. Sr-doped Zn2GeO4 persistent luminescence nanorods (ZGO:Sr NRs) served as detection probes, which exhibited blue emission. The decay times and intensity of luminescence of ZGO:Sr NRs were optimized to acquire desired luminescence properties. An aptamer-guided ratiometric persistent luminescence sensor with the LOD (0.46 pg mL-1) was established to detect carcinoembryonic antigen (CEA). This developed ratiometric aptasensor based on persistent luminescence nanomaterials (PLMs) does not only use the afterglow properties of nanomaterials to avoid the interference of autofluorescence but also precludes the interference of certain factors in the detection environment on the luminescence intensity due to the introduction of a reference signal, and is suitable for early screening of tumor markers in serum samples. Moreover, the optimization of luminescence properties, especially for luminescence decay times, provides a way for the fabrication of multiple persistent luminescence materials in the application of time-resolved fluorescence technology. Graphical abstract Construction of ZGO:Sr NR- and ZGC@PDA NP-driven ratiometric aptasensor for CEA detection.

H. pylori infection alters repair of DNA double-strand breaks via SNHG17.

Chronic infections can lead to carcinogenesis through inflammation-related mechanisms. Chronic infection of the human gastric mucosa with Helicobacter pylori is a well-known risk factor for gastric cancer. However, the mechanisms underlying H. pylori-induced gastric carcinogenesis are incompletely defined. We aimed to screen and clarify the functions of long noncoding RNAs (lncRNAs) that are differentially expressed in H. pylori-related gastric cancer. We found that lncRNA SNHG17 was upregulated by H. pylori infection and markedly increased the levels of double-strand breaks (DSBs). SNHG17 overexpression correlated with poor overall survival in patients with gastric cancer. The recruitment of NONO by overabundant nuclear SNHG17, along with the role of cytoplasmic SNHG17 as a decoy for miR-3909, which regulates Rad51 expression, shifted the DSB repair balance from homologous recombination toward nonhomologous end joining. Notably, during chronic H. pylori infection, SNHG17 knockdown inhibited chromosomal aberrations. Our findings suggest that spatially independent deregulation of the SNHG17/NONO and SNHG17/miR-3909/RING1/Rad51 pathways upon H. pylori infection promotes tumorigenesis in gastric cancer by altering the DNA repair system, which is critical for the maintenance of genomic stability. Upregulation of SNHG17 by H. pylori infection might be an undefined link between cancer and inflammation.

[Effects of Long Non-coding RNA Plasmacytoma Variant Translocation 1 Gene on Inflammatory Response and Cell Migration in Helicobacter Pylori Infected Gastric Epithelial Cell Line].

Objective To investigate the mechanism of long non-coding RNA plasmacytoma variant translocation 1 (PVT1) in gastric cancer caused by helicobacter pylori (HP) infection. Methods The expression of PVT1 was detected by quantitative real-time polymerase chain reaction in HP-infected normal gastric epithelial cells GES-1. Gastric cancer cell line SGC-7901 was transfected with PVT1 small interfering RNA and co-cultured with HP,and then the inflammatory cytokines such as tumor necrosis factor-α (TNF-α),interleukin (IL) -1ß,IL-6 and IL-8 were detected. After PVT1 was knocked down,the effects of PVT1 on the proliferation and migration of gastric cancer cells were examined by cell scratch assay. RNA-pulldown combined with mass spectrometry was used to detect the protein binding to PVT1,and the result of mass spectrometry was verified by RNA-pulldown combined with Western blot. Results In HP-infected normal gastric epithelial cells GES-1,quantitative real-time polymerase chain reaction showed that PVT1 was significantly up-regulated (t=7.160,P=0.019). PVT1 was knocked down in gastric cancer cells,and then infected with HP. The expressions of inflammatory factors including TNF-α (t=3.899,P=0.011),IL-1ß (t=14.610,P=0.000),and IL-8 (t=6.557,P=0.001) were significantly inhibited. Although PVT1 knockdown had no significant effect on the proliferation ability of gastric cancer cells,it inhibited the migration of cells. PVT1 might interact with RPS8 protein. Conclusion PVT1 may act as a pro-inflammatory factor and regulate gastric cancer caused by HP infection.

Au Film-Au@Ag Core-Shell Nanoparticle Structured Surface-Enhanced Raman Spectroscopy Aptasensor for Accurate Ochratoxin A Detection.

As a powerful toxin that could cause fatal death, the detection of ochratoxin A (OTA) has gained much attention in the fields of environmental and food sciences. In this study, an internal standard (IS) aptasensor was synthesized through a facile and scalable method to enhance the sensitivity and quantativity of OTA detection. The substrates were formed through hybridization of modified aptamers on Au@Ag core-shell nanoparticles (NPs) and Au films at a silicon surface. Incorporated with 4-ATP and 4-NTP as an internal standard, OTA recognition of such aptamers could cause NP release and signal losses. Utilizing the strong peaks at 1078 and 1335 cm-1, which represent 4-ATP and 4-NTP, respectively, the intensity ratio of I1078/I1335 could delegate the OTA concentration in a ratiometric manner. Therefore, the highest ratio of I1078/I1335 represents the lowest concentration of OTA, and a lower ratio means a higher OTA concentration. Quantitatively, the high consistency for OTA detection was achieved through correction of signal losses by IS references with an R2 of 0.993 and RSD of 0.94%, and the OTA detection limit of 5 pM was achieved. Herein, such an IS aptasensor provides a reliable and scalable detection platform for various molecules in a continuous and high-throughput manner and holds great promise in future quantitative SERS measurements.

The pharmacological efficacy of the anti-IL17 scFv and sTNFR1 bispecific fusion protein in inflammation mouse stimulated by LPS.

Acute lung injury (ALI) is still a leading cause of morbidity and mortality in critically ill patients. Recently, our study found that a bispecific fusion protein treatment can ameliorate the lung injury induced by LPS. However, the molecular mechanisms which bispecific fusion protein ameliorates acute lung injury remain unclear. In this study, we found that the bispecific fusion protein treatment inhibited the nuclear transcription of NF-κB in confocal laser scanning fluorescence microscopy, the bispecific fusion protein exert protective effects in the cell model of ALI induced by lipopolysaccharide (LPS) via inhibiting the nuclear factor κB (NF-κB) signaling pathway and mediate inflammation. Moreover, the treatment of the bispecific fusion protein show its efficacy in animal models stimulated by LPS, the results of real-time PCR and ELISA demonstrate that bispecific fusion protein treatment effectively inhibited the over-expression of inflammatory cytokines(tumor necrosis factor α, interleukin 1ß and interleukin 17). In addition, LPS-challenged mice exhibited significant lung injury characterized by the deterioration of histopathology, which was meliorated by bispecific fusion protein treatment. Collectively, these results demonstrate that bispecific fusion protein treatment ameliorates LPS-induced ALI through reducing inflammatory cytokines and lung inflammation, which may be associated with the decreased the nuclear transcription of NF-κB. The bispecific fusion protein may be useful as a novel therapy to treat ALI.

Shear stress with appropriate time-step and amplification enhances endothelial cell retention on vascular grafts.

Endothelial cells (ECs) are sensitive to changes in shear stress. The application of shear stress to ECs has been well documented to improve cell retention when placed into a haemodynamically active environment. However, the relationship between the time-step and amplification of shear stress on EC functions remains elusive. In the present study, human umbilical cord veins endothelial cells (HUVECs) were seeded on silk fibroin nanofibrous scaffolds and were preconditioned by shear stress at different time-steps and amplifications. It is shown that gradually increasing shear stress with appropriate time-steps and amplification could improve EC retention, yielding a complete endothelial-like monolayer both in vitro and in vivo. The mechanism of this improvement is mediated, at least in part, by an upregulation of integrin ß1 and focal adhesion kinase (FAK) expression, which contributed to fibronectin (FN) assembly enhancement in ECs in response to the shear stress. A modest gradual increase in shear stress was essential to allow additional time for ECs to gradually acclimatize to the changing environment, with the goal of withstanding the physiological levels of shear stress. This study recognized that the time-steps and amplifications of shear stress could regulate EC tolerance to shear stress and the anti-thrombogenicity function of engineered vascular grafts via an extracellular cell matrix-specific, mechanosensitive signalling pathway and might prevent thrombus formation in vivo. Copyright © 2016 John Wiley & Sons, Ltd.

[Professor HE Tianyou's clinical experience of acupuncture and medicine on intractable facial paralysis].

Professor HE Tianyou's unique understanding and treatment characteristics for intractahle facial paralysis are introduced. In clinical practice professor HE highly values acupoint selection and manipulation application, and integrates Chinese and western medicine to flexibly choose acupoints and formulate prescriptions according to syndrome differentiation and location differentiation, besides, he creates several specialized manipulation methods including "tug-of war opposite acupuncture method" and "tractive flash cupping". Based on strengthening body and dredging collaterals. more attention is given on stimulation to local paralyzed facial nerves; meanwhile acupuncture and medication are combined to improve clinical efficacy. During the treatment, the important role of psychological counseling on patient's anxiety is emphasized, and comprehensive treatment is given physically and psychologically in order to achieve the purpose of total rehabilitation.

Methylation of genistein and kaempferol improves their affinities for proteins.

Methylation of flavonoids appears to be a simple and effective way to improve metabolic resistance and transport of flavonoids. Serum albumins are major soluble proteins serving as transport proteins for many exogenous compounds. This work in here mainly concerns about the effect of methylation of flavonoids on the affinity for human serum albumin (HSA) and ovalbumin. One isoflavone (genistein) and one flavonol (kaempferol) and their monomethylated derivatives at position 4' (biochanin A and kaempferide) were studied for their affinities for ovalbumin and HSA. The methylation of flavonoids significantly affects the binding process. In general, the methylation of flavonoids improved the affinities for proteins by 2-16 times. This result supports that the methylation of genistein and kaempferol enhanced the transporting ability, which leads to facilitated absorption and greatly increased bioavailability. The methylation increases the hydrophobicity of genistein and kaempferol, and the hydrophobic interaction plays an important role in binding flavonoids to HSA and ovoalbumin.

Electrogenerated chemiluminescence determination of C-reactive protein with carboxyl CdSe/ZnS core/shell quantum dots.

Electrogenerated chemiluminescence (ECL) of water-soluble core/shell CdSe/ZnS quantum dots (QDs) coated with carboxylated polyethylene glycol polymers ("Qdot 625") was investigated in aqueous solutions using 2-(dibutylamino)ethanol (DBAE) and tri-n-propylamine (TPrA) as ECL coreactants. In both cases, ECL emissions at glassy carbon (GC) electrode appeared at the same potential of approximately 0.80 V vs. Ag/AgCl (3.0 M KCl), which was approximately 200 and approximately 150 mV more positive compared with the oxidation potentials for DBAE (approximately +0.60 V vs. Ag/AgCl) and TPrA (approximately +0.65 V vs. Ag/AgCl), respectively. The ECL intensity, however, was significantly affected by the type and the concentration of the ECL coreactant used as well as the nature of the working electrode. Under the present experimental conditions, ECL from DBAE was approximately 17 times stronger than that from TPrA. The maximum ECL was obtained at GC electrode when [DBAE] approximately = 53 mM, where a ratio of 11:3:1 in ECL intensity was evaluated for GC, Au, and Pt electrodes, respectively. The ECL emission of the Qdot 625/DBAE system had an apparent peak value of approximately 625 nm that matched well the fluorescence data. The QD as a label for ECL-based immunoassays of C-reactive protein (CRP) was realized by covalent binding of avidin on its surface, which allowed biotinylated anti-CRP to be attached and interacted with solution-phase CRP and the anti-CRP linked to micro-sized magnetic beads. The newly formed sandwich type aggregates were separated magnetically from the solution matrix, followed by the ECL generation at partially transparent Au nanoparticle-coated ITO electrode or Au/CD electrode in the presence of DBAE. Much stronger ECL responses were observed from the Au/CD electrode, at which a dynamic range of 1.0-10.0 microg mL(-1) CRP and a limit of detection of 1.0 microg mL(-1) CRP were obtained, respectively.