A novel pyrosequencing strategy for RHD zygosity for predicting risk of hemolytic disease of the fetus and newborn.

OBJECTIVE: The aim of this study was the development of an accurate and quantitative pyrosequence (PSQ) method for paternal RHD zygosity detection to help risk management of hemolytic disease of the fetus and newborn (HDFN). METHODS: Blood samples from 96 individuals were genotyped for RHD zygosity using pyrosequencing assay. To validate the accuracy of pyrosequencing results, all the samples were then detected by the mismatch polymerase chain reaction with sequence-specific primers (PCR-SSP) method and Sanger DNA sequencing. Serological tests were performed to assess RhD phenotypes. RESULTS: Serological results revealed that 36 cases were RhD-positive and 60 cases were RhD-negative. The concordance rate between pyrosequencing assay and mismatch PCR-SSP assay was 94.8% (91/96). There were 5 discordant results between pyrosequencing and the mismatch PCR-SSP assay. Sanger sequencing confirmed that the pyrosequencing assay correctly assigned zygosity for the 5 samples. CONCLUSION: This DNA pyrosequencing method accurately detect RHD zygosity and will help risk management of pregnancies that are at risk of HDFN.

Immune microenvironment-related gene mapping predicts immunochemotherapy response and prognosis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL). The R-CHOP immunochemotherapy regimen is the first-line treatment option for DLBCL patients and has greatly improved the prognosis of DLBCL, making it a curable disease. However, drug resistance or relapse is the main challenge for current DLBCL treatment. Studies have shown that the tumor microenvironment plays an important role in the onset, development, and responsiveness to drugs in DLBCL. Here, we used the CIBERSORT algorithm to resolve the composition of the immune microenvironment of 471 DLBCL patients from the GEO database. We found that activated memory CD4+ T cells and γδ T cells were significantly associated with immunochemotherapy response. Weighted gene co-expression networks (WGCNA) were constructed using differentially expressed genes from immunochemotherapy responders and non-responders. The module most associated with these two types of T cells was defined as hub module. Enrichment analysis of the hub module showed that baseline immune status was significantly stronger in responders than in non-responders. A protein-protein interaction (PPI) network was constructed for hub module to identify hub genes. After survival analysis, five prognosis-related genes (CD3G, CD3D, GNB4, FCHO2, GPR183) were identified and all these genes were significantly negatively associated with PD1. Using our own patient cohort, we validated the efficacy of CD3G and CD3D in predicting immunochemotherapy response. Our study showed that CD3G, CD3D, GNB4, FCHO2, and GPR183 are involved in the regulation of the immune microenvironment of DLBCL. They can be used as biomarkers for predicting immunochemotherapy response and potential therapeutic targets in DLBCL.

Preliminary study on the function of TMEM50A and its correlation with the RH genes.

OBJECTIVES: The purpose of this study was to investigate the association and impact of TMEM50A on RH genes activity and function. BACKGROUND: SMP1 is located on chromosome 1p36.11 in the RH gene locus, between the RHD and RHCE gene, where its position may be linked to RH haplotypes and contribute to selective pressures regarding certain RH haplotypes. TMEM50A is encoded by the SMP1 located in the intergenic region of RH, its influence on the function of the RH genes remains unclear. METHODS: The expression of TMEM50A was regulated by transfection of plasmid and siRNA in K562 cell model. Western blot and real-time PCR were used to detect possible expression changes in the RH. The ammonium transport function of cells was monitored using pH-sensitive dye, while transcriptome sequencing was used to predict the potential function of TMEM50A. RESULTS: The overexpression of TMEM50A significantly up-regulated RHCE gene activity (63.56%). The inhibition of TMEM50A resulted in significantly decreased RHCE (41.82%) and RHD expression (27.35%). Compared to control group, there was no significant change in the NH4 + transport function of cells in the overexpressed TMEM50A group. Transcriptome analysis showed that TMEM50A not only affected the transcription of target gene through splicing activities, but also played a role in the development of embryonic nervous system. CONCLUSIONS: TMEM50A may regulate the expression of RH gene by affecting the stability of RH mRNA through splicing function. It speculates that TMEM50A may play an important role in the development of embryonic nervous system.

Changes in Coagulation and Fibrinolysis in Post-Cesarean Section Parturients Treated With Low Molecular Weight Heparin.

BACKGROUND: Cesarean section is an independent risk factor for Venous thromboembolism (VTE). Low molecular weight heparin (LMWH) is extensively used for VTE prophylaxis after cesarean section. In this study, the effects of LMWH on coagulation and fibrinolysis after cesarean section and its clinical value were explored by studying the changes in laboratory indicators. METHODS: Antepartum and postpartum peripheral blood of 44 pregnant women who underwent vaginal delivery and 44 pregnant women who underwent cesarean section treated per routine with LMWH thromboprophylaxis on the first day post-operatively were collected for the following tests: D-dimer; thrombotic markers such as thrombomodulin (TM), thrombin-antithrombin complex (TAT), α2-plasmin inhibitor-plasmin complex (PIC), and tissue plasminogen activator inhibitor complex (t-PAIC); thromboelastography. RESULTS: Compared to the antepartum levels, PIC increased, TM, TAT, and t-PAIC decreased significantly in the parturients after a spontaneous vaginal delivery. Compared to the antepartum levels, parturients routinely treated with LMWH after cesarean section had higher PIC levels and lower D-dimer, TAT, and t-PAIC levels. Compared with parturients after vaginal delivery, parturients treated with LMWH after cesarean section had higher levels of TM, R, and MA, while there was no significant differences in the levels of D-dimer, TAT, PIC, t-PAIC, K, angle, LY30, and CI. CONCLUSION: The coagulation and fibrinolytic systems in gravidas and parturients are in a high level of dynamic equilibrium. The levels of coagulation and fibrinolytic system activation were similar in parturients who were routinely treated with LMWH after cesarean section compared with parturients after a spontaneous vaginal delivery.

Sinusoidal stimulation on afferent fibers modulates the firing pattern of downstream neurons in rat hippocampus.

Electrical stimulation in the brain is an emerging therapy for treating a wide range of neurological disorders. Although electrical pulses are commonly used in the clinic, other electrical waveforms such as sinusoidal-waves have been investigated to improve the therapeutic efficacy, to reduce the risk of tissue damage induced by stimulation, and to decrease the consumption of electrical energy. However, the effects of sinusoidal stimulation on neuronal activity are still unclear. In the present study, we investigated the neuronal responses to the stimulation of 50-Hz sinusoidal-waves applied on the afferent fibers of the neurons in the hippocampal CA1 region of Sprague-Dawley rat in vivo. Results show that the stimulation increased the firing rate of both pyramidal neurons and interneurons in the downstream region of stimulation. Also, the stimulation eliminated the original theta rhythms (2-5 Hz) in the single-unit activity of the two types of neurons and entrained these neurons to fire at the stimulation rhythm. These results provide new clues for the mechanisms of brain stimulation to suppress the pathological rhythms in the neuronal activity, and for the application of sinusoidal waveforms in brain stimulation therapy.

Effect of allogeneic blood transfusion on levels of IL-6 and sIL-R2 in peripheral blood of children with acute lymphocytic leukemia.

Effect of allogeneic blood transfusion on the expression of interleukin-6 (IL-6) and soluble interleukin-2 receptor (sIL-2R) in peripheral blood of children with acute lymphoblastic leukemia (ALL) was investigated. A total of 91 ALL children admitted to Nanfang Hospital from June 2014 to January 2017 were selected as the study group. Patients were randomly divided into allogeneic blood transfusion group (n=38) and non-transfusion group (n=53). In addition, a total of 64 healthy children were also selected from June 2014 to January 2017 as the control group. Patients in allogeneic blood transfusion group were transfused with red blood cell suspension and machine-collected platelets, while patients in non-transfusion group were not treated with blood transfusion. Peripheral venous blood was collected before and at 4, 8 and 12 weeks after blood transfusion to prepare serum. Serum IL-6 and sIL-2R levels were measured by enzyme-linked immunosorbent assay (ELISA). Before transfusion, serum levels of IL-6 and sIL-2R were significantly lower in the study group than those in control group (p<0.05), and no significant differences in serum levels of IL-6 and sIL-2R were found between the allogeneic blood transfusion and non-transfusion group. After transfusion, serum levels of IL-6 and sIL-2R were stable for 12 weeks in the non-transfusion group, while IL-6 and sIL-2R levels were significantly increased in the allogeneic blood transfusion group. The results showed that serum level of IL-6 and sIL-2R was increased in ALL patients with allogeneic blood transfusion, which resulted in reduced antibody production and decreased cellular immunity. The patients had low immunity, and attention should be paid on the pathogen infection prevention.

Intron 4 of the RH Gene in Han Chinese, Tibetan, and Mongol Populations.

AIMS: This study aimed to compare the intron 4 sequence of the RHD and RHCE genes from Han Chinese, Tibetans, and Mongols, and explore its polymorphisms. MATERIALS AND METHODS: To investigate the distinction in the RHD and RHCE intron 4, polymerase chain reaction (PCR) was performed by a set of primers: Intron4F and Intron4R. Primer Intron4F for a sequence located in exon 4 and primer Intron4R for a sequence located in exon 5, respectively. RHD and RHCE intron 4 of all the samples from 26 cases of random unrelated Hans (13 RhD-positive donors and 13 RhD-negative donors), 25 cases of random unrelated Tibetans (18 RhD-positive donors and 7 RhD-negative donors), and 4 cases of random unrelated Mongols (1 RhD-positive donor and 3 RhD-negative donors) were amplified with PCR. The PCR products were then sequenced. RESULTS: A 576-bp product was detected in all the Han, Tibetan, and Mongol RhD-positive donors, whereas a 1228-bp product was detected in RhD-negative donors. The sequences of RHCE gene intron 4 were identical to each other in all Han, Tibetan, and Mongol RhD-negative donors, including 335 bp of Alu element, with a whole length of 1078 bp. By contrast, a 426-bp product was detected in all Han, Tibetan, and Mongol RhD-positive donors. Compared with the RHCE gene, a 652-bp deletion was noted in the RHD gene of Chinese, including the whole Alu element. The results were similar to the findings of Caucasians, whereas the lengths of RHD gene deletion fragments of Japanese and French were 649 and 654 or 651 bp, respectively. CONCLUSIONS: The RHCE gene intron 4 of Han Chinese, Tibetans, and Mongols differs from the RHD gene intron 4 in the presence of a 652-bp fragment. The RHCE gene intron 4 in Chinese has its own structural characteristics and differs among various ethnicities and regions.