Identification and Blood Transfusion of a Rare A1.02/BA.02 Genotype.

BACKGROUND: The aim was to analyze the serological and molecular genetic characteristics of a rare B(A) subtype pedigree, explore its pathogenesis, and discuss transfusion strategies. METHODS: ABO blood typing serological tests were conducted on a female subject and her family member using standard serological methods. Sequencing analysis of the ABO gene exons 6 and 7 was performed using PCR technique for the female subject and her family member to examine the blood types of the participants. RESULTS: The serological test results showed a discrepancy between the forward and reverse typings of the female subject. The forward typing was similar to that of AB subtype serological forward typing, while the reverse typing indicated AB blood type. Based on the sequencing results, it is inferred that the female subject and her son have 8 mutations on one BA.02 chain: 297A>G, 526C>G, 657C>T, 700C>G, 703G>A, 796C>A, 803G>C, and 930G>A. Comparing these eight mutation sites with the Blood Group Antigen Gene Mutation Database (BGMUT), it was found that the female subject had a heterozygous mutation at c.700C>G in the 7th exon of the B.01 gene, consistent with the characteristics of the BA.02 allele. The genotype of the female subject was determined as A1.02/ BA.02, while the genotype of her son was determined as O.01.01/BA.02. CONCLUSIONS: The serological presentation of the B(A) subtype for the female subject reported in this study was unique. It differed from previously reported cases, indicating that the determination of B(A) subtypes cannot solely rely on serological testing. It requires a comprehensive analysis combining the results of genetic testing and pedigree investigation.

Molecular Genetic Analysis of Para-Bombay Phenotype in Chinese: Identification of a Novel FUT1 Allele.

BACKGROUND: The goal was to identify a novel FUT1 allele and to study serologic and gene feature of the para-Bombay blood type of one expectant mother in Xinjiang, China. METHODS: ABO and Lewis groups were recognized by standard serologic techniques in an ABO typing discrepancy specimen from one person at the Tianjin Blood Center. DNA (deoxyribonucleic acid) was collected and polymerase chain reactions with sequence-specific primers (PCR-SSP) were performed to sequence exons 6 and 7 of ABO gene, exon 4 of FUT1 gene, and exon 2 of FUT2. PCR products were sequenced to identify ABO groups and the variation sites. The genotype was determined by family study. RESULTS: In our laboratory testing, erythrocytes from the proposita did not react with anti-A and anti-B reagents. B antigen was discovered only after adsorption and elution. Red cells were nonreactive with monoclonal anti-H. The sera of the proposita contained anti-A and were weakly agglutinated by B cells. The hybrid 902 A>G mutation was detected in the proposita's father and mother. The proposita has the same mutation 902 A>G, which was conjectured as homozygosity for 902 A>G. CONCLUSIONS: One novel mutation of FUT1 gene was observed in our laboratory. It has never been reported previously. The para-Bombay phenotype in the proposita originating from Xinjiang (China) results from homozygosity for FUT1 902 A>G, together with 357 C>T of FUT2.

Rh Phenotype and Allele Frequencies Among 88,856 Patients in China.

BACKGROUND: This study was performed to provide information on the frequencies of Rh antigens, alleles, and phenotypes from our region in Tianjin, China. METHODS: This observational study was conducted on patients from January 2018 to March 2021 using a fully automated system for ABO and Rh typing of blood cells. The phenotypes of C, c, E, and e were detected by the slide method. The data were collected and calculations done to determine the antigen, phenotypes and allele frequencies. RESULTS: Four hundred thirty-three cases of Rh (D) negative phenotype were confirmed in 88,856 patients. Of the four Rh antigens (C, c, E, e) that were phenotyped by serological methods, the "e" antigen was found to have the highest frequency (99.74%). The most common Rh negative phenotype observed was ccdee, followed by Ccdee. The prevalence of Rh phenotypes ccdEe, CCdee, CcdEe, CCdEe, ccdEE were found to be rare in our population with percentages of 0.0473%, 0.018%, 0.018%, 0.0034%, and 0.0011%, respectively. CONCLUSIONS: Knowledge of red cell antigen phenotype frequencies in a population is helpful in terms of their ethnic distribution. We have determined the prevalence of Rh antigens and Rh phenotypes in China. The Rh blood group distribution in this population was different from that in other populations.

Identification and Blood Transfusion of Rare B(A) Blood Group.

BACKGROUND: To study serologic and gene characteristics of the B(A) blood group of one patient in Tianjin, China. To investigate the blood transfusion for patients with B(A) blood group. METHODS: ABO subgroups were identified by standard serologic techniques in ABO typing discrepancy sample from one patient at the Tianjin Medical University General Hospital. DNA (deoxyribonucleic acid) was collected from his sample and PCR (polymerase chain reaction) was used to sequence exons 6 and 7. PCR products were sequenced to identify ABO subgroups and the B(A) allele. Molecular genotyping of the ABO gene was performed by DNA sequencing of exons 6 and 7 of the ABO gene. RESULTS: The patient's red cells showed mixed field agglutination reaction (MF) with anti-A and weak reaction (W+) with anti-H. However, it has strong agglutination reaction with anti-B. The patient's serum showed the presence of anti-A antibody, while it can't react with B cell. The serological characteristics of the patient's red cells were similar to AxB subtype. Variation sites were confirmed at 261delG, 297 A>G, 526 C>G, 640 A>G, 657 C>T, 703 G>A, 796 C>A, 803 G>C, 930 G>A in exon 6 and 7 of the ABO gene. The sequence was similar to B101 except for nt640 (A>G). Genotyping indicated that the specimen was B(A)04. CONCLUSIONS: Molecular genotyping confirmed the ABO status as B(A)04. The B(A) blood group is associated with a complicated serologic phenotype and DNA detection is necessary for this atypical phenotype sample. To ensure the safety of transfusion, this study developed an emergency transfusion procedure for patients with B(A) type.

A standardized quantitative method for detecting remnant alpha-Gal antigen in animal tissues or animal tissue-derived biomaterials and its application.

Alpha-Gal (Gal) epitopes present in animal tissues are known to be the key xenoantigens that elicit xenorejection. However, a standardized method to determine Gal epitope in animal tissue-derived biomaterials does not exist. Herein, a standardized method for quantitative detection of Gal antigen was established based on an ELISA inhibition assay with Gal antibody. In this method, the key optimized experimental conditions were: (1) Gal-antigen positive and negative reference materials were developed, and used as positive and negative control in the test system, respectively; (2) A mixture of artificial Gal-BSA antigen plus Gal-negative matrix was used as the calibration standard sample, making it has similar composition with test sample; and (3) The lysis buffer was combined with the homogenate to expose the Gal antigen as much as possible. The results from validation and application experiments showed that the standardized method had good reproducibility (RSD = 12.48%), and the lower detection limit (LDL) is ~7.1 × 1011 Gal epitopes/reaction. This method has been further developed into a detection Kit (Meitan 70101, China), and it has been developed as a standard method for detecting remnant immunogen of animal tissue derived medical devices, and as the industry standard has been released in China. (YY/T 1561-2017).

[Assessment Method of Remnant α-1, 3-galactosyle Epitopes in Animal Tissue-derived Biomaterials].

The aim of this study was to establish an assessment method for determining α-Gal (α-1, 3-galactosyle) epitopes contained in animal tissue or animal tissue-derived biological materials with ELISA inhibition assay. Firstly, a 96 well plate was coated with Gal α-1, 3-Gal/bovine serum albumin (BSA) as a solid phase antigen and meanwhile, the anti-α-Gal M86 was used to react with α-Gal antigens which contained in the test materials. Then, the residual antibodies (M86) in the supernatant of M86-Gal reaction mixture were measured using ELISA inhibition assay by the α-Gal coating plate. The inhibition curve of the ELISA inhibition assay, the R2 = 0.999, was well established. Checking using both α-Gal positive materials (rat liver tissues) and α-Gal negative materials (human placenta tissues) showed a good sensitivity and specificity. Based on the presently established method, the α-Gal expression profile of rat tissues, decellular animal tissue-derived biological materials and porcine dermal before and after decellular treatment were determined. The M86 ELISA inhibition assay method, which can quantitatively determine the α-Gal antigens contained in animal tissues or animal tissue-derived biomaterials, was refined. This M86 specific antibody based-ELISA inhibition assay established in the present study has good sensitivity and specificity, and could be a useful method for determining remnant α-1, 3Gal antigens in animal tissue-derived biomaterials.

Tetrandrine potentiates the hypoglycemic efficacy of berberine by inhibiting P-glycoprotein function.

This study was designed to improve the absorption and hypoglycemic efficacy of berberine (BBR), which is a substrate of P-glycoprotein (P-gp), by combination with a P-gp inhibitor tetrandrine (Tet). Flow cytometry and LC-MS/MS were used to determine the cellular efflux or retention of chemicals. Pharmacokinetic study was performed in ICR mice following oral administration of the study compounds. The hypoglycemic efficacies of the compounds were evaluated in diabetic KK-Ay mice. In the in vitro experiments, Tet significantly inhibited the efflux and increased the uptake of P-gp substrates rhodamine-123 as well as BBR in MCF7/DOX cells and Caco-2 intestinal cells. Meanwhile, Tet greatly reduced the expression of P-gp in Caco-2 cells. The inhibition of BBR efflux by Tet was translated into improved pharmacokinetics in vivo. When co-administered, Tet dose-dependently increased the average maximum concentration (C(max)) and area under concentration-time curve (AUC0₋24) of BBR in mice. Tet itself had no impact on glucose metabolism. However, it greatly potentiated the hypoglycemic efficacy of BBR in diabetic KK-Ay mice. In addition, we found that Tet had moderate inhibitory effect on the catalytic activity of CYP3A4, which played a role in the bio-transformation of BBR, and this may also take part in the improvement of the pharmacokinetics of BBR. In summary, combination with P-gp inhibitors such as Tet can improve the pharmacokinetics and hypoglycemic efficacy of BBR greatly; this implicates a feasible strategy for exploring the therapeutic effects of BBR and other pharmaceuticals which are substrates of P-gp.

Berberine analogue IMB-Y53 improves glucose-lowering efficacy by averting cellular efflux especially P-glycoprotein efflux.

OBJECTIVE: Cellular efflux transporters, especially P-glycoprotein (P-gp), impel berberine (BBR) out of cells, and therefore reduce bioavailability of the compound. This study was designed to overcome efflux of BBR using P-gp as a target. MATERIALS/METHODS: Molecular docking study was done to identify BBR analogues that were with low affinity to P-gp. Flow cytometry was used to determine cellular efflux of chemicals. Pharmacokinetic study was performed in Wistar rats, following oral administration of the study compounds. The efficacies of chemicals on glucose homeostasis were determined both in cultured cells and diabetic KK-Ay and db/db mice. RESULTS: In the molecular docking study, we found that among BBR analogues pseudo-berberine (IMB-Y53) has low affinity to P-gp. IMB-Y53 was retained in Caco-2, HL-7702 and C2C12 cells for a significantly longer period of time than BBR did. P-gp inhibitor tetrandrine (Tet) abolished the efflux of BBR at different extent depending on the expression level of P-gp; however, Tet had no impact on IMB-Y53 efflux. BBR increased P-gp expression dose-dependently in intestinal and liver cells; IMB-Y53 also up-regulated P-gp but at a much lower level as compared with BBR. Administered at equal dose in rats, the maximum plasma concentration (C(max)) and area under concentration-time curve (AUC(0-24)) of IMB-Y53 were 1.61 and 2.27-fold of those of BBR, respectively, indicating an improved bioavailability. IMB-Y53 stimulated glucose utility in cultured cells with a degree similar to that of BBR, but exhibited enhanced glucose-lowering efficacy in KK-Ay and db/db diabetic mice. CONCLUSIONS: These results suggest that overcoming cellular efflux especially P-gp's function improves bioavailability and hypoglycemic effect of BBR.

Synthesis and structure-activity relationship of berberine analogues in LDLR up-regulation and AMPK activation.

Currently there is no approved medicine for the treatment of metabolic syndrome. A series of new derivatives of berberine (BBR) or pseudoberberine (1) was synthesized and evaluated for their activity on AMP-activated protein kinase (AMPK) activation and up-regulatory low-density-lipoprotein receptor (LDLR) gene expression, respectively. In addition, the four major metabolites of BBR in vivo were also examined for their activity on AMPK in order to further understand the chemical mechanisms responsible for its glucose-lowering efficacy. Among those BBR analogues, compound 1 exhibited the potential effect on AMPK activation and LDLR up-regulation as compared with BBR. The results suggested that compound 1 might be a multiple-target agent for the treatment of metabolic syndrome, and thus was selected as a promising drug candidate for further development.

Host apolipoprotein B messenger RNA-editing enzyme catalytic polypeptide-like 3G is an innate defensive factor and drug target against hepatitis C virus.

UNLABELLED: Host cellular factor apolipoprotein B messenger RNA (mRNA)-editing enzyme catalytic polypeptide-like 3G (hA3G) is a cytidine deaminase that inhibits a group of viruses including human immunodeficiency virus-1 (HIV-1). In the continuation of our research on hA3G, we found that hA3G stabilizing compounds significantly inhibited hepatitis C virus (HCV) replication. Therefore, this study investigated the role of hA3G in HCV replication. Introduction of external hA3G into HCV-infected Huh7.5 human hepatocytes inhibited HCV replication; knockdown of endogenous hA3G enhanced HCV replication. Exogenous HIV-1 virion infectivity factor (Vif) decreased intracellular hA3G and therefore enhanced HCV proliferation, suggesting that the presence of Vif might be an explanation for the HIV-1/HCV coinfection often observed in HIV-1(+) individuals. Treatment of the HCV-infected Huh7.5 cells with RN-5 or IMB-26, two known hA3G stabilizing compounds, increased intracellular hA3G and accordingly inhibited HCV replication. The compounds inhibit HCV through increasing the level of hA3G incorporated into HCV particles, but not through inhibiting HCV enzymes. However, G/A hypermutation in the HCV genome were not detected, suggesting a new antiviral mechanism of hA3G in HCV, different from that in HIV-1. Stabilization of hA3G by RN-5 was safe in vivo. CONCLUSION: hA3G appears to be a cellular restrict factor against HCV and could be a potential target for drug discovery.

Synthesis and structure-activity relationship of N-(2-arylethyl) isoquinoline derivatives as human scavenger receptor CD36 antagonists.

By using human scavenger receptor CD36 as the target, twenty-five N-(2-arylethyl) isoquinoline derivatives were designed, synthesized and evaluated for their antagonistic activities for CD36-oxidatively low density lipoprotein (oxLDL) binding. The primary analysis of structure-activity relationship (SAR) indicated a methoxyl at the 7-position and a hydroxyl at the 6- or 8-position could afford good activities. Among these analogs, compounds 7e and 7t showed the potential CD36 antagonistic activities with IC(50) values of 0.2 and 0.8 µg/mL, respectively. Furthermore, both of them could effectively inhibit oxLDL uptake in insect Sf9 cells overexpressing human CD36, and thus have been selected for further investigation. We consider N-(2-arylethyl) isoquinoline analogs to be a family of novel CD36 antagonists.

Design and synthesis of oxymatrine analogues overcoming drug resistance in hepatitis B virus through targeting host heat stress cognate 70.

Heat-stress cognate 70 (Hsc70) is a host protein required for hepatitis B virus (HBV) replication, and oxymatrine (1) suppresses Hsc70 expression. Taking Hsc70 as a target against HBV, 22 analogues of 1 defined with substituents at position 1, 13, or 14 were synthesized and evaluated for their activity on Hsc70 mRNA expression. The SAR revealed that (i) the oxygen atom at the 1-position was not essential, (ii) increasing electron density on the ring D reduced the activity, and (iii) introducing a proper substituent at the 13- and/or 14-position(s), especially electron-withdrawing groups, might enhance the activity. Among the analogues, 6b possessing 13-ethoxyl afforded an increased activity in respect to 1. Importantly, it was active for either wild-type or lamivudine-resistant HBV, as its target is host Hsc70 but not viral enzymes. LD(50) of 6b in mice was over 750 mg/kg in oral route. We consider compound 6b promising for further investigation.

Protein kinase D activation stimulates the transcription of the insulin receptor gene.

Our previous studies proved that berberine (BBR) up-regulates the insulin receptor (InsR) gene by stimulating its promoter and calphostin C blocks this effect. Here, the present study was designed to discover the specific kinase isoform(s) used by berberine. In the blocking experiment, we found that Gö6976, a kinase inhibitor that potently inhibit PKCµ/protein kinase D 1 (PKD1), effectively and specifically reduced the activity of BBR on InsR. PKD1/PKCµ is a member of the PKD family that also covers PKD2 and PKD3/PKCν with high homology. The role of PKD1 in InsR expression was also proved by using another PKD-activator oligomycin. In the RNA interference experiment, we found that the effects of BBR on InsR expression and on cellular glucose consumption were partially eliminated by silencing any one of the three PKDs and were totally abolished by silencing all of them. BBR enhanced the PKD1 catalytic activity, but not its expression. Along with BBR treatment, PKD1 ser916 autophosphorylation was increased time- and dose-dependently, indicating an activation of PKD1 by BBR. BBR also induces PKD1 translocation from cytosol-to-plasma membrane, further verifying the activation of PKD1. These results suggest that the PKD family is involved in the transcriptional regulation of the InsR gene; we consider it to be a potential new target to discover drugs for sugar-related disorders in the future.

Comprehensive analysis of sequence-specific stability of siRNA.

Double-stranded small interfering RNAs (siRNAs) are important modulators of biological processes and hold great promise for therapeutic applications. However, serum processing of synthetic siRNAs is still largely unknown. To address this issue, serum degradation assays of 125 siRNAs were first performed in this study. Four siRNA categories of distinct serum stability were identified, including a group of siRNAs that were stable in their native form for both in vitro and in vivo assays. Fine mapping of the cleavage events occurring in serum treatment demonstrated that most occurred at two vulnerable sites, leading to a speculation that rational modification of these sites might protect most siRNAs from serum degradation. For proof of concept, an exhaustive siRNA modification study was performed. In addition to the consistent stabilization pattern revealed at these sites, our study further showed that a single modification made at the cleavage site stabilized the siRNAs to a large extent, highlighting the importance of these sites in siRNA degradation. In summary, the present study provided a comprehensive picture of serum processing of siRNA as well as a starting point for a rational siRNA modification strategy, both of which are of great importance to in vivo and therapeutic applications of siRNA.

[Effects of basic fibroblast growth factor on protein kinase B activity and c-fos expression in CNE- I nasopharyngeal carcinoma cell line].

OBJECTIVE: The relationship between basic fibroblast growth factor (bFGF) and phosphatidylinositol 3-kinase/protein kinase B(PI 3-K/PKB) signal pathway was studied during the course of bFGF regulating carcinoma epithelioid cell lines- I (CNE- I ) nasopharyngeal carcinoma cell proliferation through altering time of bFGF treatment. METHODS: PKB activities were measured by advanced Takai method. C-fos expression was examined by Western Blot. RESULTS: bFGF (50 ng/ml) significantly stimulated PKB activity,After pretreatment of Wortmannin for 1 h, the PKB activity decreases to the basal levels(P < 0. 05),compared with control group. After treatment of bFGF (50 ng/ml) at various time, the expression of c-fos is increased. The expression of c-fos increases to the peak when the time of bFGF treatment is 30 min. After pretreatment of Wortmannin for 1 h, the expression of c-fos decreases 20% when the time of bFGF treatment was 30 min and when the time of bFGF treatment was 45 min, the expression of c-fos decreases 35%. CONCLUSIONS: 1. PI 3-K and PKB mediate bFGF-induced signal transduction in CNE- I nasopharyngeal carcinoma cell line; PKB lies in downstream of PI 3-K. 2. bFGF induces the increased expression of c-fos through PI 3-K/PKB in CNE- I nasopharyngeal carcinoma cell line.