Molecular and Family Analyses of a Novel RHD1058G>C Allele in a Chinese RhD Population.

BACKGROUND: Rh(D) phenotype in a sample from a 19-year-old female patient showed weak positivity (1+). A follow-up sample was requested to further define the Rh(D) phenotype, her Rh(D) phenotype was tested by using another reagent, Rh(D) phenotype still showed weak reactivity (1+), RhCcEe phenotype was Ccee. METHODS: Seven samples from the family members of the proposita were received. The RhDCcEe phenotypes were typed by the microcolumn gel card and the unexpected antibodies were assayed by indirect anti-human globulin test (IAT). Genomic DNA was extracted from the blood sample and the novel RHD1058G>C allele was detected through an established sequence-specific primer PCR (PCR-SSP), RHD exons 1 - 10 were sequenced afterward by exon-specific amplification. The distribution of RHD1058G>C allele and RHD weak positive phenotype were investigated in the pedigrees. RESULTS: The unexpected antibodies all were negative in the family members. The novel RHD1058G>C allele was found in the proposita, her father, and grandfather. Five family members were detected serologically with the common Rh(D)-positive phenotypes either as homozygote of RHD/RHD or heterozygote of RHD/RHd. Two family members were detected as weak D phenotypes in accordance with the genotyping results by PCR-SSP, and both of them have a D1058Ce haplotype and a dce haplotype. One member, her father, was tested common Rh(D)-positive with D1058Ce haplotype and a Dce haplotype. CONCLUSIONS: These data allow us to describe the characteristics of the weak D phenotype with a novel c.RHD-1058G>C allele, which may be partial D and increase the risk of RHD alloantibody. The novel RHD1058G>C allele was inherited in three generations in a family rather than spontaneous mutation in an individual.

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

BACKGROUND: Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. OBJECTIVES: The aim of this study was to explore a possible pathogenic role of DON in FK. METHODS: We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. RESULTS: The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. CONCLUSIONS: DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

A Rare Heterozygote with a Novel IVS-â ¡-786 (T>A) Mutation on ß-Globin Gene in a Patient with Thalassemia.

BACKGROUND: Thalassemia is an inherited hemolytic blood disease, whose pathogenesis is an imbalance in the expression of hemoglobin. We report a case of a rare ß-globin gene intron mutation for thalassemia patient. METHODS: The blood routine test was performed with an automatic blood cell analyzer. Hb analysis was conducted by hemoglobin (Hb) analyzer. The common ß-thalassemia and α-thalassemia gene mutations were detected by Gap-PCR and fluorescence PCR melting curve, and the rare ß-thalassemia gene mutations were detected by DNA sequencing. RESULTS: A rare heterozygous mutation of ß-globin gene IVS-II-786 (T>A) was found in this case. Blood routine analysis showed the following values: Hb 92 g/L, RBC 4.1 x 1012/L, MCV 74.10 fL, MCH 22.4 pg, MCHC 303 g/L, HCT 0.304 L/L, and RET-He 22.7 pg. Hemoglobin analysis showed values of HbA2 2.2% and HbF < 2% by automatic capillary electrophoresis. The results of gene analysis and DNA sequencing showed that the ß-globin gene IVS-II-786 (T>A) mutation was heterozygous. CONCLUSIONS: The heterozygote of ß-globin gene IVS-II-786 (T>A) mutation was detected for the first time, and the clinical manifestation was moderate anemia. Hemoglobin analysis indicated that the level of HbA2 was decreased. This mutation is relatively rare and easy to misdiagnose in clinical practice. It will provide a new type of evidence and guidance for genetic counseling and clinical treatment of beta thalassemia.

A Novel RHCE*cE (c.827C>A) Allele, Containing the Single-Nucleotide Change, Encodes Altered c/E Antigens.

The RH blood group system is the most complex with over 50 antigens. So far over hundreds of RhCE variant alleles have been described resulting in weakened and/or partial expression of RhCE antigens [1], some variant Rh phenotypes are caused by exchange of genetic material between the RHD and RHCE genes, resulting in many hybrid genes, other phenotypes result from missense mutations. Variant alleles encode altered phenotypes with either weakened antigens, lacked antigens, or unexpected antigens. Besides, the mutation of RH blood group genes may lead to the changes of Rh antigen epitopes. RHCE gene mutations or polymorphisms may bring about altered RH antigens in quality and quantity [2]. Serologic weaknesses or discrepancies are regularly faced by blood transfusion laboratories, and molecular background explaining this feature can be precisely characterized only by the molecular biological methods.

Effects of exogenous indole-3-acetic acid on the density of trichomes, expression of artemisinin biosynthetic genes, and artemisinin biosynthesis in Artemisia annua.

Artemisinin is the most practical medication for the treatment of malaria, but is only very minimally synthesized in Artemisia annua, significantly less than the market needs. In this study, indole-3-acetic acid (IAA) was used to investigate its effects on trichomes, artemisinin accumulation, and biosynthetic gene expression in A. anuua. The results showed that exogenous IAA could contribute to the growth and development of A. annua and increase the density of trichomes. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that artemisinin and dihydroartemisinic acid (DHAA) contents were increased by 1.9-fold (1.1 mg/g) and 2.1-fold (0.51 mg/g) after IAA treatment in comparison with control lines (CK), respectively. Furthermore, quantitative real-time PCR results showed that AaADS, AaCYP71AV1, AaALDH1, and AaDBR2, four critical enzyme genes for the biosynthesis of artemisinin, had relatively high transcription levels in leaves of A. annua treated with IAA. In summary, this study indicated that exogenous IAA treatment was a feasible strategy to enhance artemisinin production, which paves the way for further metabolic engineering of artemisinin biosynthesis.

Rational design of porous starch/hyaluronic acid composites for hemostasis.

Effective hemorrhage control is pivotal for decreasing the trauma death both in civilian and military but has proven to be dauntingly challenging, especially for solid viscera and artery trauma. Here we report the fabrication of a novel starch-based hemostat, sodium trimethaphosphate (STMP)-crosslinked starch/hyaluronic acid (HA) (ScSH) porous composites. Aiming at hemostatic potential, physicochemical properties, cytocompatibility, hemocompatibility, histocompatibility and hemostatic performance of ScSH composites have been studied. As it turned out, the incorporation of HA greatly improved the water absorption capacity and hemostatic performance of ScSH composites. In addition, the composites with a non-toxic crosslinker exhibited non-cytotoxicity, low hemolysis ratio (0.97%) and favorable histocompatibility. Meanwhile, the composites performed exceptionally well in blood clotting of superficial injury, solid viscera and artery trauma and displayed similar hemostatic efficacy to commercialized hemostat (Quickclean® particles). Unambiguously, these encouraging results highlighted potential of our materials to be used as hemostats and made the approach, constructing porous starch/HA composites, a promising strategy to accelerate further development of hemostatic agents applied both in vivo and in vitro.

[Identification of RhCcEe Mixed Visual Field in Patients with Regular Blood Transfusion and Efficacy Analysis of the Matched Transfusion].

OBJECTIVE: To explore the feasibility of RhCcEe blood group antigen mixed visual field identification in patients with regular blood transfusion, to follow up and evaluate the efficacy of matched transfusion and its clinical significance. METHODS: RhCcEe genotyping for 142 patients with regular transfusion in our hospital was carried out by PCR-SSP method. According to the results of genotyping, 48 patients voluntarily selected the continuous transfusion of RhCcEe matched red blood cells, 46 patients received random blood transfusion (RhCcEe mismatched transfusion), 42 patients received partial RhCcEe matched transfusion (unable to provide fully matched RhCcEe donors each time), and 6 patients' blood transfusion data were lost. After 3-6 months of the RhCcEe matched transfusion, all patients were tested by RhCcEe microcolumn gel card and compared with the results before RhCcEe matched transfusion. The positive rates of alloantibodies, DAT and the percentage of red blood cell invalid transfusion were followed up and evaluated for the above-mentsioned 3 types of regular transfusion patients in the past 5 years. RESULTS: Out of the 48 patients who underwent conti-nuous RhCcEe matched transfusion, only 1 case showed stratification, the remaining 47 cases had clear gel card results without stratification, suggesting that PCR-SSP genotyping was feasible. In addition, another 42 patients who could not receive RhCcEe matched transfusion each time and 46 patients with random blood transfusion were found to have a mixed vision phenomenon again. but the results was still difficult to confirm the results. For the transfusion results in the past 5 years, follow-up analysis showed that there were 1 case alloantibody (anti-Jka) (1/48) , 1 case of DAT positive (1/48) and 2 cases of invalid transfusion (2/48) in the RhCcEe matched transfusion group; 7 cases of alloantibodies (3 anti-E, 1 anti-E+anti-c, 1 anti-C, 1 anti-M, 1 anti-Fya) (7/46), 6 case of DAT positive (6/46) and 9 case of invalid transfusion (9/46) in the random transfusion group; 6 cases of alloantibodies (1 anti-E, 1 anti-E+autoantibody, 1 anti-C, 1 anti-c, 1 anti-M and 1 other antibody) (6/42) and 7 case of DAT positive (7/42) and 8 case of invalid transfusion (8/42) in the partial RhCcEe matched transfusion group. The statistical analysis showed that the positive rate of alloantibodies and the invalid infusion rate of RBC in each group were significant differences between RhCcEe matched transfusion group and the random transfusion group as well as betwen Rhce fe matched transfusion group and the partial matched transfusion group（P＜0.05）, but there was no statistical difference between the random transfusion group and the partial matched transfusion group（P>0.05）. CONCLUSION: PCR-SSP genotyping technique can be used to detect RhCcEe mixed vision in patients with regular blood transfusion. Continuous RhCcEe matched transfusion can effectively prevent the occurrence of alloimmunization, and improve the clinical transfusion efficacy and safety of the patients with regular blood transfusion, which has very important clinical significance.

[Screening and Identification of Blood Group Alloantibody by Surface Plasmon Resonance Technique and Its Preliminary Application].

OBJECTIVE: To investigate the feasibilily of screening and identifying the red blood cell type alloantibodies by means of surface plasman resonance(SPR) technique so as to provide a new method for detecting the transfusion compatibility of red blood cells. METHODS: The RBC antigens for screening the alloantibody were fixed on the SPR chip surface by means of amino coupling method; the analysis conditions of SPR chip were optimized and then the control serum with RBC blood group antibody positive was detected; the performance of SPR chip for detection of serum was analysed; the consistance of rusults detected by SPR technique and microcolum agglutination for clinieal samples of 129 thalasstmia patients with history of lone-term blood transfusion were compared; at the same time, the blood group amtibodies in 7 patients with blood group antibody positive were identified before blood transfusion by using SPR chip so as to select the RBC antigen compatible blood for transfusion; and the efficacy of RBC transfusion was followed up and evaluated. RESULTS: The repeatability, sensitivity and specificity of SPR chip technique for detecting the blood group alloantibodies all were better. The SPR technique and microcolumn agglutination method were not significant different for screening blood group alloantibodies (χ2 = 0.333, P＞0.05), and the overall consistency was 97.2%; the results of SPR technique in 7 patients with positive blood group antibodies were as follows: 3 cases with anti-E, 1 case anti-M, 1 case anti-C, 1 case anti-Jka and 1 case autoantibody, which were consistent with the results of microcolumn agglutination tests, and the compatible red blood cells were selected for transfusion, of which the infusion of 6 cases was effective. In only 1 case the infusion was ineffective because of autoantibody. CONCLUSION: For screening and identification of blood group alloantibodies, the performance of SPR chip technique is equivalent to the micro-column agglutination, but the procedure of SPR technique is simpler, faster and high-throughput and label-free, which can meet the basic requirements for rapid screening and identification of blood group alloantibodies before transfusion of red blood cells.

Surface plasmons excited by multiple layer grating.

The surface plasmons that are excited by the multiple layer grating structures on the gold thin film are studied using the finite-difference time-domain method in this paper. The structure parameters' effects on the coupling enhancement of surface plasmons are examined, and the structure design guidelines are given. It is found that the distance between the grating layers and the distance between the gratings and gold thin film are the key structure parameters for better cavity resonances. To have the stronger field enhancements of the excited surface plasmons for the multilayer grating structures, it is found that the width of the gratings should be smaller for the lower grating layers. The multiple layer gratings with proper structure designs can have better performances than single layer grating structure because the cavity effects can enhance the light coupling and more light can be coupled into the surface plasmons by more layers of grating. It is found that the maximum electric field intensity for five layer grating structures can be 163% of the case of the single layer grating structure in our simulations.

Rh-Matched Transfusion through Molecular Typing for ß-Thalassemia Patients Is Required and Feasible in Chinese.

BACKGROUND: Molecular typing for RHCE blood group alleles has been established in many countries for patients and blood donors. In the Chinese literature nearly 80% of transfused patients with alloimmunization have antibodies specific for antigens of the Rh blood group system. We investigated if it is feasible to match packed red blood cells (RBCs) for Chinese ß-thalassemia patients by RHCE genotyping. METHODS: In this study, 481 patients with ß-thalassemia were enrolled. They were genotyped for RHCE alleles by a simple PCR method with sequence-specific primers (PCR-SSP). Among these patients, 203 continuously received RBCs of the identical Rh subgroups according to the genotyping results for at least 3 months. Subsequently, their phenotypes were tested through a micro-column gel card method. For validation purposes, 400 donors were serologically typed with the same technology, of which 164 were genotyped too. Finally, the C, c, E, and e frequencies and the feasibility of the simple genotyping method were analyzed. RESULTS: All patients showed mixed-field agglutination in the Rh subgroup gel cards before the same Rh subgroups in blood donors were selected for blood transfusion. The results, however, lacked mixed-field agglutination in all 203 cases after transfusion with RBC concentrates selected for the patient's C, c, E, and e antigens for at least 3 months. The genotyping results of 164 donors were all consistent with the serological results. Whole coding regions of RHCE were sequenced in 7 individuals with weak c, E, or e antigens. In only one sample we observed a 1059G>A nucleotide mutation coding for a truncated RhCE polypeptide (GenBank KT957625), in the other 6 samples no sequence variant was found. Both patients and donors were predominantly CcEe and CCee, with a prevalence of 55.3% and 24.9% for patients or 49.3% and 31.3% for donors, respectively. It revealed that about 80% of Chinese could receive Rh-matched RBCs easily. CONCLUSION: A simple RHCE genotyping technique is safe enough for Rh-matched transfusion of ß-thalassemia patients in Chinese Han.

Development of a Strategy Based on the Surface Plasmon Resonance Technology for Platelet Compatibility Testing.

BACKGROUND: This study was aimed to establish a novel strategy based on the surface plasmon resonance (SPR) technology for platelet compatibility testing. METHODS: A novel surface matrix was prepared based on poly (OEGMA-co-HEMA) via surface-initiated polymerization as a biosensor surface platform. Type O universal platelets and donor platelets were immobilized on these novel matrices via amine-coupling reaction and worked as a capturing ligand for binding the platelet antibody. Antibodies binding to platelets were monitored in real time by injecting the samples into a microfluidic channel. Clinical serum samples (n = 186) with multiple platelet transfusions were assayed for platelet antibodies using the SPR technology and monoclonal antibody-immobilized platelet antigen (MAIPA) assay. RESULTS: The novel biosensor surface achieved nonfouling background and high immobilization capacity and showed good repeatability and stability after regeneration. The limit of detection of the SPR biosensor for platelet antibody was estimated to be 50 ng/mL. The sensitivity and specificity were 92% and 98.7%. It could detect the platelet antibody directly in serum samples, and the results were similar to MAIPA assay. CONCLUSIONS: A novel strategy to facilitate the sensitive and reliable detection of platelet compatibility for developing an SPR-based biosensor was established in this study. The SPR-based biosensor combined with novel surface chemistry is a promising method for platelet compatibility testing.

Linking arsenite- and cadmium-generated oxidative stress to microsatellite instability in vitro and in vivo.

Mismatch repair (MMR) corrects replicative errors and minimizes DNA damage that occurs frequently in microsatellites. MMR deficiency is manifested as microsatellite instability (MSI), which contributes to hypermutability and cancer pathogenesis. Genomic instability, including MSI and chromosomal instability, appears to be responsible for the carcinogenesis of arsenic and cadmium, common contaminants in our environment. However, few studies have addressed arsenic- or cadmium-induced MSI, especially its potential link with arsenic- or cadmium-generated oxidative stress, due to the lack of quantifiable MSI assays and cost-effective animal models. Here, using a dual-fluorescent reporter, we demonstrate that sub-lethal doses of cadmium or arsenite, but not arsenate, increased the MSI frequency in human colorectal cancer cells. Arsenite- and cadmium-induced MSI occurred concomitantly with increased levels of reactive species and oxidative DNA damage, and with decreased levels of MMR proteins. However, N-acetyl-l-cysteine (NAC) suppressed arsenite- and cadmium-induced MSI and oxidative stress while restoring the levels of MMR proteins in the cells. Similarly, MSI was induced separately by arsenite and cadmium, and suppressed by NAC, in zebrafish in a fluorescinated PCR-based assay with newly-developed microsatellite markers and inter-segmental comparisons. Of five selected antioxidants examined, differential effects were exerted on the MSI induction and cytotoxicity of both arsenite and cadmium. Compared to MMR-proficient cells, MMR-deficient cells were more resistant to arsenic-mediated and cadmium-mediated cytotoxicity. Our findings demonstrate a novel linkage between arsenite-generated and cadmium-generated oxidative stress and MSI induction. Our findings also caution that antioxidants must be individually validated before being used for preventing arsenite- and cadmium-induced MSI that is associated with cancer development.

[RH Factor and Clinical Transfusion Effectiveness for ß-Thalassemia Children with Long-Term Blood Transfusion].

OBJECTIVE: To investigate the irregular antibody production and its relationship with Rh factor genotypes and the loci of thalassemia gene mutations for the ß-thalassemic children with long-term transfusion, so as provide experimental basis for clinical safe and effective transfusions for thalassemic children. METHODS: The peripheral blood from 246 children with ß-thalassemia was collected in our hospital; the extraction of genomic DNA and Rh factor (C/c, E/e) genotypes were assayed by PCR-SSP method, the irregular antibodies were screened and identified by serological method, the genotypes for thalassemia and gene mutations were analysed by PCR-RD method. RESULTS: The genotypes of Rh factors classified by PCR- SSP in the 246 cases of ß-thalassemia children were as follws: Ce/Ce (143/246, 58.1%), CE/ce (59/246, 24%), cE/cE (14/24, 5.7%), Ce/ce (12/246, 4.9%); The positive rate of irregular antibody was 7.7% (19/246), including anti-E (7/19), anti-c (5/19), anti-C (2/19), anti-E and anti-c (2/19), anti-e (1/19), anti-D (2/19); Of the 19 cases with positive irregular antibody, the genotypings of Rh factor were: Ce/Ce (11/19), CE/ce (2/19), cE/cE (2/19), Ce/ce (2/19), cE/ce (2/19); the gene mutations location of thalassemia for 19 cases with positive irregular antibody: CD41-42M (13/19), CD71-72M (2/19), IVS-II-654M (3/19), -28M (1/19). CONCLUSION: The irregular antibody production for ß-thalassemic children with long-term transfusion may have some relevance with Rh factor genotypes and thalassemia genetic mutations. This study possesses a certain significance for effective prevention of RBC alloimmune response of ß-thalassemia children and improvement of efficacy and safety of clinical transfasion blood.

Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses.

A surface plasmon resonance (SPR)-based biosensor was developed for specific detection of nine common respiratory virus, including influenza A and influenza B, H1N1, respiratory syncytial virus (RSV), parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and severe acute respiratory syndrome coronavirus (SARS). The SPR biosensor was developed by immobilizing nine respiratory virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin was used to label the PCR primer and further amplify the signal by introducing streptavidin after hybridization. Throat swab specimens representing nine common respiratory viruses were tested by the innovative SPR-based biosensor to evaluate the sensitivity, specificity and reproducibility of this method. Results suggest that this biosensor has the potential to simultaneously identify common respiratory viruses.