Suspension Array Research Articles

A unique ex vivo tumor model: 3D cocultured system with cancer and stromal cells including blood microvessels.

211 Background: Importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and tolerance against treatment. Although 2D culture and spheroid consisting only cancer cells still remains the preferred platform for most laboratory preclinical studies while these provide only limited information about tumor microenvironment. In order to mimic the patient tumor tissue, ex vivo model which recaptures the tumor microenvironment is required. Methods: Layered 3D stromal tissues comprising microvascular network were produced by culturing fibroblasts and endothelial cells coated with the extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and co-cultured tumor were morphologically evaluated by HE stain, immunohistochemistry and immunofluorescence (IF). Their gene expression and secretome profile were characterized by RNA-sequencing and bio-plex suspension array technologies. Furthermore, drug sensitivity assay were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of JFCR. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Results: The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and the tendency that dense microvascular network was formed nearby cancer cells was observed. In comparison with 2D culture or 3D mono-cultured spheroid model, decreased drug sensitivities were represented in the layered 3D co-cultured model. Omics profiles difference among models suggest that our 3D model has some similarity to in vivo tumor. Conclusions: We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug sensitivity in the co-cultured tumors may reflect the response of cancer cells in in vivo. Our unique 3D ex vivo model appear to be a valuable tool for drug evaluation, and thus testing approved and/or developing compounds with patient-derived cells would enable better prediction their efficacy.

Production of insulin-like growth factor-binding proteins during the development of hepatic fibrosis due to chronic hepatitis C

Introduction and AimsInsulin-like growth factor 1 is modulated by the insulin-like growth factor-binding proteins (IGFBPs) that are synthesized in the liver. The aim of the present study was to evaluate the concentrations of IGFBPs 1–7 in patients with chronic hepatitis C and study their association with fibrosis stage. Patients and methodsA prospective, cross-sectional study was conducted that included patients with chronic hepatitis C. The stages of fibrosis were determined through FibroTest and FibroScan and the patients were compared with a control group. Serum levels of IGFBPs 1–7 were quantified through multiple suspension arrays. The Kruskal-Wallis test, Mann-Whitney U test, Spearman’s correlation, and ROC curves were used for the statistical analysis. ResultsUpon comparing the patients and controls, the highest concentrations were found in IGFBPs 1, 2, 4, and 7 (p=0.02, 0.002, 0.008, and <0.001, respectively). IGFBP-3 levels had a tendency to be lower in the patients (p=0.066), whereas values were similar between patients and controls for IGFBP-5 and 6 (p=0.786 and p=0.244, respectively). Of the seven IGFBPs, IGFBP-3 concentrations were the highest. There were significant differences between fibrosis stages for IGFBP-5 and IGFBP-7. ConclusionIGFBPs play a relevant role in the fibrotic process in liver damage. IGFBP-7, in particular, differentiates fibrosis stages, making it a potential serum biomarker.

Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

BackgroundMalaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries.ResultsThe qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density.ConclusionThis immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

Bone Marrow Plasma Cytokine Signature Profiles in Severe Aplastic Anemia.

Objective We studied bone marrow plasma (BMP) cytokines in severe aplastic anemia (SAA) patients and healthy volunteers to investigate differences in the cytokine profiles between them and propose a cytokine signature of SAA. Methods A Bio-Plex suspension array system was used to measure 27 analytes in BMP samples from 47 SAA patients and 30 healthy donors. Results Compared to healthy people, SAA patients had higher levels of tumor necrosis factor α (TNF-α (TNF-γ (IFN-γ (IFN-β (MIP-1β (MIP-1α (TNF-α (TNF-β (MIP-1β (MIP-1β (MIP-1γ (IFN-α (TNF-Conclusions The current study demonstrated distinct cytokine profiles among untreated SAA patients, recovering SAA (RSAA) patients, and healthy people. The cytokines of RSAA patients showed similar characteristics to those of untreated SAA patients and healthy people, respectively, which may reflect that the immune status of RSAA patients is in different stages of recovery after IST; thus, it may provide an important tool in diagnosing and evaluating or predicting curative effects in clinics.

Rapid detection of CYP2C9, CYP2C19,CYP4F2,VKORC1 and ABCB1 gene polymorphisms by liquid phase chip technology

Objective To establish a method for simultaneous and rapid detecting of the polymorphisms in Cytochrome P450 2C9 (CYP2C9), CYP2C19, CYP4F2, Vitamin K epoxide reductase (VKORC1) and ATP-binding cassette subfamily B member1 (ABCB1) gene, which were associated with warfarin and clopidogrel, based on liquid phase chip technology. Methods Method establishment. The eight gene sequences near targeted sites related to warfarin and clopidogrel were found in Genbank, and the specific primers and probes were designed. Through multiple PCR amplification, followed by allele specific primer extension (ASPE), and MagPlex-Tag microspheres hybridization, the suspension array Luminex 200 system step-by-step, the genotypes were determined by fluorescence signal. The reaction system was optimized and its methodological evaluation was performed. 260 patients with antithrombotic therapy from Dongguan houjie hospital were recruited in this study form June 2017 to December 2018. The eight genotypes of the 260 patients were detected by the established method, and the results were compared with the sequencing results. Results The results of 260 samples showed that allelic median fluorescence intensity (MFI) ratios of homozygotes (mutant/wild-type) were all greater than 0.9 or less than 0.1, and all the allelic MFI ratios of heterozygotes were between 0.3 and 0.6. The within run and between run coefficients of variance for allelic MFI ratios were lower than 6.4% and 10.9%, respectively. The minimum DNA template requirements was 0.75ng. The genotypes of 260 patients determined by the established method were completely concordant with the sequencing results. Conclusion A method was established successfully for rapid detecting the genotypes which associated with warfarin and clopidogrel based on liquid phase chip technology. Key words: Liquid phase chip technology; Allele-specific primer extension; Warfarin; Clopidogrel; Single nucleotide polymorphism

Optical Thickness-Encoded Suspension Array for High-Throughput Multiplexed Gene Detection.

We proposed a coding and decoding method of suspension array (SA) based on micro-quartz pieces (MQPs) with different optical thicknesses. The capture probes (cDNA) were grafted onto the surfaces of MQPs and specifically recognized and combined with the partial sequence of the target DNA (tDNA) to form a MQP-cDNA-tDNA complex. Quantum dot-labeled signal probes were then used to specifically recognize and bind another portion of the tDNA in the complex to form a double-probe sandwich structure. This optical thickness-encoded SA can be decoded and detected by a dual-wavelength digital holographic phase fluorescence microscope system. We conducted a series of DNA molecule detection experiments by using this encoding method. Control experiments confirmed the specificity of optical thickness-encoded SA in DNA detection. The concentration gradient experiments then demonstrated the response of the MQPs based SA to analyte concentration. Finally, we used the encoding method to detect three types of DNA in a single sample and confirmed the feasibility of the proposed optical thickness-encoded SA in multiplexed DNA detection. The detection results are stable, and the detection exhibits high specificity and good repeatability.

PH-responsive polyaniline/polyethylene glycol composite arrays for colorimetric sensor application

Polyanilines are conjugated polymers that exhibit dynamic optical and electrical properties upon pH change. In this study, we constructed two different types of polyaniline/polyethylene glycol (PANI/PEG) composite arrays via simple PEG photolithography: (1) positional PANI/PEG array films and (2) non-positional PANI/PEG suspension arrays. The patterning process based on PEG photolithography allows the precise control of shapes and sizes in PANI/PEG composite arrays. Both positional/non-positional PANI/PEG composite arrays demonstrated reversible optical property changes accompanied by excellent colorimetric changes from deep blue to blue, cyan, and green in response to pH changes. Specifically, we used PANI/PEG composite arrays as a pH sensitive indicator to detect bacterial growth because of their colorimetric responses to pH changes. The PANI/PEG composite arrays exhibited distinct changes in the two absorption bands at 420 and 600 nm with a color change when exposed directly to Escherichia coli (E. coli) growth medium. We envision that the novel approach described here could provide a new insight into the design and construction of versatile optical, electrochemical, electronic sensors based on functional conjugated polymer/hydrogel composite materials.

Production of insulin-like growth factor-binding proteins during the development of hepatic fibrosis due to chronic hepatitis C.

Insulin-like growth factor 1 is modulated by the insulin-like growth factor-binding proteins (IGFBPs) that are synthesized in the liver. The aim of the present study was to evaluate the concentrations of IGFBPs 1-7 in patients with chronic hepatitis C and study their association with fibrosis stage. A prospective, cross-sectional study was conducted that included patients with chronic hepatitis C. The stages of fibrosis were determined through FibroTest and FibroScan and the patients were compared with a control group. Serum levels of IGFBPs 1-7 were quantified through multiple suspension arrays. The Kruskal-Wallis test, Mann-Whitney U test, Spearman's correlation, and ROC curves were used for the statistical analysis. Upon comparing the patients and controls, the highest concentrations were found in IGFBPs 1, 2, 4, and 7 (p=0.02, p=0.002, p=0.008, and p<0.001, respectively). IGFBP-3 levels had a tendency to be lower in the patients (p=0.066), whereas values were similar between patients and controls for IGFBP-5 and 6 (p=0.786 and p=0.244, respectively). Of the seven IGFBPs, IGFBP-3 concentrations were the highest. There were significant differences between fibrosis stages for IGFBP-5 and IGFBP-7. IGFBPs play a relevant role in the fibrotic process in liver damage. IGFBP-7, in particular, differentiates fibrosis stages, making it a potential serum biomarker.

Granzyme A Participates in the Pathogenesis of Infection-Associated Acute Encephalopathy.

The present study aimed to determine whether granzymes are implicated in the pathogenesis of infection-associated acute encephalopathy (AE). We investigated granzyme and cytokine levels in the cerebrospinal fluid of patients with acute encephalopathy or complex febrile seizures (cFS). A total of 24 acute encephalopathy patients and 22 complex febrile seizures patients were included in the present study. Levels of granzymes A and B were measured using enzyme-linked immunosorbent assay, and levels of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin 1β (IL-1β), IL-1 receptor antagonist (IL-1RA), IL-4, IL-6, IL-8, and IL-10 were assessed using the Bio-Plex suspension array system. Cerebrospinal fluid levels of granzyme A were significantly higher, and those of TNF-α and IL-1RA were significantly lower in the AE group than in the cFS group; however, no significant differences in the levels of granzyme B, IFN-γ, IL-1β, IL-4, IL-6, IL-8, and IL-10 were observed between the 2 groups. In addition, no significant differences in granzyme A, granzyme B, or cytokine levels were observed between acute encephalopathy patients with and those without neurologic sequelae. Our findings indicate the involvement of granzyme A in the pathogenesis of acute encephalopathy.

Change of cytokines after intravitreal ranibizumab in patients with recurrent branch retinal vein occlusion and macular edema.

To investigate the relations of vascular endothelial growth factor, growth factors, soluble vascular endothelial growth factor receptors, and inflammatory factors to recurrence of macular edema after anti-vascular endothelial growth factor therapy in patients with branch retinal vein occlusion. This study retrospectively investigated 17 patients with branch retinal vein occlusion who received intravitreal ranibizumab injection three times within 6 months for recurrent macular edema. Aqueous humor samples were obtained from these patients at every recurrence. Levels of soluble vascular endothelial growth factor receptor-1, soluble vascular endothelial growth factor receptor-2, vascular endothelial growth factor, placental growth factor, platelet-derived growth factor-AA, soluble intercellular adhesion molecule-1, monocyte chemoattractant protein-1, interleukin-6, interleukin-8, interleukin-12(p70), and interleukin-13 were measured by the suspension array method. Aqueous flare values were measured with a laser flare meter and central macular thickness was determined by optical coherence tomography. Mean best-corrected visual acuity and central macular thickness improved significantly over time after intravitreal ranibizumab injection, but the aqueous flare value at recurrence after intravitreal ranibizumab injection showed no significant change compared with baseline. Aqueous humor levels of soluble vascular endothelial growth factor receptor-1, soluble vascular endothelial growth factor receptor-2, vascular endothelial growth factor, platelet-derived growth factor-AA, monocyte chemoattractant protein-1, and interleukin-8 decreased significantly over time after intravitreal ranibizumab injection. However, there were no significant changes of the other five factors/cytokines (placental growth factor, soluble intercellular adhesion molecule-1, interleukin-6, interleukin-12, and interleukin-13) at recurrence after intravitreal ranibizumab injection compared with baseline. These findings suggest that persistent inflammation may influence the recurrence of macular edema in branch retinal vein occlusion patients, and that adding steroid therapy might be an effective strategy for preventing recurrence.

Precisely Encoded Barcodes Using Tetrapod CdSe/CdS Quantum Dots with a Large Stokes Shift for Multiplexed Detection

AbstractA serious obstacle to the construction of high‐capacity optical barcodes in suspension array technology is energy transfer, which can prompt unpredictable barcode signals, limited barcode numbers, and the need for an unfeasible number of experimental iterations. This work reports an effective and simple way to eliminate energy transfer in multicolor quantum dots (QDs)‐encoded microbeads by incorporating tetrapod CdSe/CdS QDs with a large Stokes shift (about 180 nm). Exploiting this unique feature enables the facile realization of a theoretical 7 × 7‐1 barcoding matrix combining two colors and seven intensity levels. As such, microbeads containing tetrapod CdSe/CdS QDs are demonstrated to possess a powerful encoding capacity which allows for precise barcode design. The ability of the Shirasu porous glass membrane emulsification method to easily control microbead size facilitates the establishment of a 3D barcode library of 144 distinguishable barcodes, indicating the enormous potential to enable large‐scale multiplexed detection. Moreover, when applied for the multiplexed detection of five common allergens, these barcodes exhibit superior detection performance (limit of detection: 0.01–0.02 IU mL−1) for both spiked and patient serum samples. Therefore, this new coding strategy helps to expand barcoding capacity while simultaneously reducing the technical and economic barriers to the optical encoding of microbeads for high‐throughput multiplexed detection.

VAR2CSA Serology to Detect Plasmodium falciparum Transmission Patterns in Pregnancy.

Pregnant women constitute a promising sentinel group for continuous monitoring of malaria transmission. To identify antibody signatures of recent Plasmodium falciparum exposure during pregnancy, we dissected IgG responses against VAR2CSA, the parasite antigen that mediates placental sequestration. We used a multiplex peptide-based suspension array in 2,354 samples from pregnant women from Mozambique, Benin, Kenya, Gabon, Tanzania, and Spain. Two VAR2CSA peptides of limited polymorphism were immunogenic and targeted by IgG responses readily boosted during infection and with estimated half-lives of <2 years. Seroprevalence against these peptides reflected declines and rebounds of transmission in southern Mozambique during 2004–2012, reduced exposure associated with use of preventive measures during pregnancy, and local clusters of transmission that were missed by detection of P. falciparum infections. These data suggest that VAR2CSA serology can provide a useful adjunct for the fine-scale estimation of the malaria burden among pregnant women over time and space.

Characterizing Salmonella enterica Serovar Choleraesuis, var. Kunzendorf: A Comparative Case Study.

Different Salmonella serovars generally display different antigenic formulae, but there are some exceptions. For instance, the same antigenic formula, 6,7:c:1,5, is shared by Salmonella enterica serovar, Paratyphi C, Typhisuis, and Choleraesuis. Moreover, three biotypes have been described within the S. Choleraesuis serovar. A distinction among such biotypes can only be based on biochemical behaviors (biotyping) posing serious concerns when rapid characterization is required. The study of an outbreak of severe epizootic salmonellosis in wild boars occurred in Italy between 2012 and 2014 and the typing of the isolates recovered from the outbreak were used to test different approaches for serovar identification. A number of 30 S. Choleraesuis var. Kunzendorf isolates from the outbreak were typed by means of four different methods to derive serovar and biotype: (i) slide agglutination method followed by biochemical tests, (ii) suspension array xMAP® Salmonella Serotyping Assay (SSA), (iii) whole genome sequencing (WGS) and data analysis using SeqSero tool, and (iv) WGS and data analysis using Salmonella TypeFinder tool. Slide agglutination, xMAP® SSA and WGS, followed by SeqSero analysis, are methods that infer the serovars according to the White-Kauffmann-Le Minor (WKL) scheme, based exclusively on antigens. Using these methods, isolates with incomplete antigenic formulae could be misleadingly excluded from an outbreak. On the contrary, WGS followed by Salmonella TypeFinder data analysis, which predicts the serotype on the basis of Multilocus sequence typing (MLST), might be able to cluster together isolates belonging to the same outbreak irrespective of the antigenic formula. Results suggest the benefit of routine use of a combination of in silico MLST and antigenic formula analysis to solve specific ambiguous case studies for outbreak investigation purposes.

Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater.

Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers. Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer. For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.

Tokishakuyakusan, a Kampo medicine, attenuates endometriosis-like lesions and hyperalgesia in murine with endometriosis-like symptoms.

How are the effects of Tokishakuyakusan (TSS), a traditional Japanese medicine (Kampo) on murine endometriosis model? BALB/c mice were used for making the murine endometriosis model. Homogeneous uterus was surgically implanted with lipopolysaccharide (LPS) in peritoneal cavity. We administered 2weeks of TSS (1.0g/kg) orally. Upon treatment completion, we performed the hot plate test for all mice and collected blood samples before sacrifice. Then, the endometriosis-like lesions and uteri in the abdominal cavity were harvested. Concentrations of several cytokines in sera and cyst fluids were measured using Bio-Plex Suspension Array System. IL-33 localization was determined by immunohistochemistry. Gene expression of inflammatory cytokines in the endometriosis-like lesions or the eutopic endometrium was evaluated by real-time RT-PCR. After 14days of TSS treatment, the numbers of endometriosis-like cysts and cyst weight were significantly decreased. In TSS-treated mice, the latency against heat stimuli was extended. Inflammatory cytokine concentrations in sera were not changed by TSS treatment. TSS intake decreased IL-33 mRNA expression in endometriosis-like lesions and led to the tendency of attenuation of the elevated IL-33 synthesis in the cyst fluids of lesions. These results suggest the TSS ameliorated the hyperalgesia and lesion formation on the LPS-accelerated endometriosis-like model. TSS represents a possible ideal target of novel therapeutics for endometriosis patients with dysmenorrhea.

Developing a novel molecular serotyping system based on capsular polysaccharide synthesis gene clusters of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a major food-borne pathogen. V. parahaemolyticus infections are associated with various serotypes; to date, 71 K-serogroups of V. parahaemolyticus have been determined based on capsular polysaccharide (CPS) diversity. In this study, the capsular polysaccharide gene clusters (CPSgcs) of 55 K-serogroups were identified by whole-genome sequencing and analysis. These CPSgcs exhibit a high level of genetic diversity. A microsphere-based suspension array (MSA) was established for the detection and identification of 55 V. parahaemolyticus K-serogroups based on CPSgc-specific genes. To evaluate our array, a double-blind test with 120 clinical isolates was carried out. In addition, an in silico K-serotyping system was established based on V. parahaemolyticus CPSgc-specific genes. This system was then used to examine 845 publicly available V. parahaemolyticus genomes; the results demonstrated that 813 isolates belong to one of 43 K-serogroups. Taken together, these results demonstrate that the molecular system developed in this study is suitable for rapid serotyping of V. parahaemolyticus isolates from environmental and clinical samples. In addition, the system could be applied to epidemiological investigations of this important food-borne pathogen.

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study

BackgroundVaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity.MethodsWe evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection.ResultsRTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3–0.93], p = 0.03, for MSP1; 0.52 [0.26–0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses.ConclusionsIncreased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

Suspension array-based deafness genetic screening in 53,033 Chinese newborns identifies high prevalence of 109 G>A in GJB2

ObjectivesMore than 50% of congenital hearing loss is attributed to genetic factors. Data of gene mutation associated with hearing loss from large population studies in Chinese population are scarce. In this study, we conducted a comprehensive newborn genetic screening in China to establish the carrier frequency and mutation spectrum of deafness-associated genes. MethodsA total of 53,033 newborns were screened for hearing defects associated mutations. Twenty hot spot mutations in GJB2, GJB3, SLC26A4 and mitochondria12S rRNA were examined using suspension array analysis. Results14,185 newborns (26.75%) were identified with at least one mutated allele. 872 (1.64%) neonates carried homozygous mutations including 112 (0.21%) mitochondrial DNA homoplasmy, 228 (0.43%) were compound heterozygotes, and 11,985 (22.59%) were heterozygotes including 11 (0.02%) mitochondrial DNA heteroplasmy. Top five mutations included 109 G > A, 235 delC, 299–300 delAT in GJB2, IVS7-2 A > G in SLC26A4 and 1555 A > G in mitochondria12S rRNA. Notably, a total of 10,995 neonates (20.73%) carried 109 G > A in GJB2. Moreover, the allele frequencies of 109 G > A were detected 11.61% in Guangdong, 10.44% in Sichuan and 2.88% in Shandong, respectively, a significant difference in prevalence among these geographic regions (p<0.01). In addition, the high frequency of 109 G > A in GJB2 was confirmed by a TaqMan probe-based qPCR assay. Very recently, the ClinGen Hearing Loss Expert Panel reached a consensus and confirmed its pathogenic role in hearing impairment. ConclusionWe delineated the mutation profile of common deafness-causing genes in the Chinese population and highlighted the high prevalence of 109 G > A pathogenic mutation. Our study may facilitate early diagnosis/intervention and genetic counseling for hearing impairment in clinical practice.

Role of Cytokines in Ranibizumab Therapy for Macular Edema in Patients with Central Retinal Vein Occlusion.

Purpose: To investigate aqueous humor levels of 11 factors/cytokines in patients with central retinal vein occlusion (CRVO) and macular edema (ME) receiving anti-vascular endothelial growth factor (VEGF) therapy, as well as correlations between changes of functional or morphological parameters and aqueous cytokine levels. Methods: In 32 CRVO patients scheduled to receive 2 consecutive doses of intravitreal ranibizumab, aqueous samples were obtained at the time of injecting each dose. Aqueous levels of VEGF, soluble VEGF receptor (sVEGFR)-1, sVEGFR-2, platelet-derived growth factor-AA (PDGF-AA), placental growth factor (PlGF), interleukin-6, and monocyte chemotactic protein-1 (MCP-1) were measured using a suspension array. Results: Aqueous humor levels of VEGF, sVEGFR-1, PDGF, PlGF, interleukin-6, and MCP-1 were all significantly lower at 1 month after the initial dose of intravitreal ranibizumab compared with baseline. A significant negative correlation was noted between the change of ME and the changes of aqueous humor VEGF or interleukin-6 levels after intravitreal ranibizumab. The change of VEGF also showed a significant negative correlation with improvement of visual acuity. Conclusions: In patients with CRVO, the changes of visual acuity and ME after intravitreal ranibizumab were associated with inhibition of intraocular VEGF production. VEGF could be a useful marker for the response of ME to treatment.

Biomarkers of prehypertension and hypertension in women

The percentage of undiagnosed prehypertensive and hypertensive women are high in the population of Kazakhstan. Our study assessed biomarkers in prehypertension and hypertension in women. 203 women were divided into 3 groups: “pre-hypertensive” group ( n = 30, 46.6 ± 6.2 y.o.) with a systolic blood pressure from 120 to 139 mmHg or/and a diastolic blood pressure from 80 to 89 mm Hg. In the “hypertensive” group the blood pressure was > = 140/90 mmHg ( n = 73, 46.6 ± 5.6 y.o.). In the normotensive group the blood pressure was < = 120/80 mm Hg ( n = 100, 44.2 ± 5.9 y.o.). The MILLIPLEX MAP Human CVD 1 Magnetic Bead kit was used for the quantification of cardiovascular biomarkers in venous samples. The quantification of the level of cardiac markers was carried out on the Bio-Plex®3D multiplex analyzer Suspension Array System (Bio-Rad Laboratories, Inc., USA). The values of brain natriuretic peptide (BNP), creatine kinase-MB (CK MB), and leptin were statistically increased in women depending on their blood pressure. The BNP values in the hypertensive group (Me = 157.9 pg/ml, Q 1 = 53.75 pg/ml and Q 3 = 229.6 pg/ml) were statistically greater (p = 0.016) than in the normotensive group (Me = 85.14 pg/ml, Q 1 = 18.55 pg/ml and Q 3 = 113.9 pg/ml). The BNP values in the prehypertension group were greater than in the normotensive group and less than in the hypertensive group (Me = 73.00 pg/ml, Q 1 = 35.52 pg/ml and Q 3 = 139.4 pg/ml). The CK MB values in the hypertensive group (Me = 3693 pg/ml, Q 1 = 3105 pg/ml and Q 3 = 6178 pg/ml) were statistically higher (p = 0.022) than in the normotensive group (Me = 2688 pg/ml, Q 1 = 1629 pg/ml and Q 3 = 4532 pg/ml). The CK MB values in the prehypertension group were greater than in the normotensive group and less than in the hypertensive group (Me = 2722 pg/ml, Q 1 = 1833 pg/ml and Q 3 = 5266 pg/ml). In addition, the values of leptin in the hypertensive group (Me = 23910 pg/ml, Q 1 = 14510 pg/ml and Q 3 = 54824 pg/ml) were statistically greater (p = 0.001) than in the normotensive group (Me = 18256 pg/ml, Q 1 = 9287 pg/ml and Q 3 = 34843 pg/ml). The leptin values in the prehypertension group were: Me = 26424 pg/ml, Q 1 = 10514 pg/ml and Q 3 = 53060 pg/ml. In hypertensive women, serum BNP, CK-MB and leptin levels were significantly different from the same parameters in control women. Pre-hypertensive patients were in-between the normotensive and the hypertensive groups. However, biomarkers were not statistically different in women with prehypertension and those from the two other groups. Thus, patients with pre-hypertension cannot longer be attributed to healthy individuals although, they have not yet pronounced clinical and laboratory signs of arterial hypertension, the mechanisms of arterial hypertension are already running. Our results showed that there is need to develop recommendations for the identification and monitoring of patients with pre-hypertension in order to prevent the development of irreversible cardiovascular alterations.