Abstract: At present, nucleic acid detection technology based on the PCR principle is commonly used to detect malaria parasites, the existing Plasmodium detection methods mainly include microscopy, antigen immunoassay, and nucleic acid detection,but due to the long detection time, high personnel and equipment requirements, and other shortcomings, its popularization, and application at the grassroots level are limited. What challenges previous Plasmodium detection methods are the lack of experienced professionals and advanced equipment at the grassroots as well as the requirement of rapid detection of large samples under extreme conditions. The isothermal amplification technology developed in recent years has potential application prospects due to its simplicity, rapidity, high sensitivity, and high specificity. This article attempts to review the principles, characteristics, and prospects of various isothermal amplification technologies, and on this basis, focuses on the introduction of recombinase polymerase amplification (RPA) and recombinase⁃aided isothermal amplification (RAA) assay technologies and proposes the use of such recombinant enzyme amplification technologies to achieve rapid and accurate diagnosis of common Plasmodium species possibility and imagination.

OBJECTIVETo compare the effect of citric acid stimulation on salivary alpha-amylase (sAA), total protein (TP), salivary flow rate, and pH value between Pi deficiency (PD) children and healthy children, thereby providing evidence for Pi controlling saliva theory.

METHODSTwenty PD children were recruited, and 29 healthy children were also recruited at the same time. Saliva samples from all subjects were collected before and after citric acid stimulation. The sAA activity and amount, TP contents, salivary flow rate, and pH value were determined and compared.

RESULTS(1) Citric acid stimulation was able to significantly increase salivary flow rate, pH value, sAA activities, sAA specific activity and sAA amount (including glycosylated and non-glycosylated sAA amount) in healthy children (P<0.05), while it could markedly increase salivary flow rate, pH value, and glycosylated sAA levels in PD children (P<0.05); (2) Although there was no statistical difference in determined salivary indices between the two groups (P>0.05), salivary indices except salivary flow rate and glycosylated sAA levels decreased more in PD children. There was statistical difference in sAA activity ratio, sAA specific activity ratio, and the ratio of glycosylated sAA levels between PD children and healthy children (P<0.05).

CONCLUSIONPD children had decreased response to citric acid stimulation.

Child ; Citric Acid ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; Saliva ; Salivary alpha-Amylases ; metabolism ; alpha-Amylases

Starting from the thinking characteristics of TCM in the holistic approach of syndrome and process of syndrome differentiation, the problems in the criteria of syndrome diagnosis was analyzed, combining with the outcome of investigation on macrocosmic diagnosis of syndrome carried on twice by expert group nationwide in China, a concept for designing basic framework of standard for macrocosmic diagnosis of syndrome was advocated in this study. In order to recognize and master fully the standard, the following items would be the contents of the framework: give a standardized term of each syndrome; the basic clinical features of a syndrome; the qualitative parameters which could be used to distinguish the nature of a syndrome, and indexes to be used to judge the affected location of a syndrome during identifying process for a syndrome; the order of pertinent indexes with their given score, and the threshold for quantitative diagnosis. It is considered that the above framework defined the sufficient criteria which is constructed on the bases of the approaches with integrated qualitative and quantitative methods. It may enable the syndrome differentiation to access the real world of a patient to be used appropriately this framework.
Diagnosis, Differential ; Humans ; Medicine, Chinese Traditional ; standards ; Syndrome ; Yang Deficiency ; diagnosis ; Yin Deficiency ; diagnosis

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology

OBJECTIVETo explore the best program for treatment of irritable bowel syndrome (IBS) of constipation type.

METHODSNinety-five cases of IBS were randomly divided into 3 groups. Group A (n = 30) were treated by acupuncture combined with microorganism pharmaceutical preparations, group B (n = 35) by oral administration of medicine for loosening the bowel to relieve constipation plus microorganism pharmaceutical preparations, and group C (n = 30) by simple acupuncture.

RESULTSThe total effective rates were 90.0%, 77.2% and 66.7%, in the group A, B and C, respectively, with a very significant differences as the group A compared with those in the groups B, C (P < 0.01), and with no significant difference as the group B compared with that of the group C (P > 0. 05). The intestinal available bacteria, bilidobacteria and lactobacillus, increased and enteric bacilli decreased in varying degrees in the 3 groups.

CONCLUSIONAcupuncture combined with microorganism pharmaceutical preparations has a better therapeutic effect on irritable bowel syndrome of constipation type.

Acupuncture Therapy ; Adult ; Combined Modality Therapy ; Constipation ; therapy ; Female ; Humans ; Intestines ; microbiology ; Irritable Bowel Syndrome ; microbiology ; therapy ; Male ; Probiotics ; therapeutic use

·AIM: To investigate the relationship between the thickness of choroid and the development of idiopathic macular epiretinal membrane (IMEM). ·METHODS: A retrospective analysis on 48 cases (48 eyes) of idiopathic macular epiretinal membrane patients (the IMEM group) was taken in our hospital from January 2014 to December 2016, and 50 right eyes in 50 healthy persons with physical examination were selected the control group, comparison on subfoveal choroidal thickness (SFCT) levels of sicked eyes, normal eyes in IMEM group and the control group were made, postoperative SFCT level change of sicked eyes in IMEM patients and normal eyes were investigated via follow-up, and analysis on correlation between postoperative choroidal thickness and the best corrected visual acuity was taken. ·RESULTS: The SFCT of sicked eyes in IMEM group 362.22±40.75μm was significantly lower than that of the contralateral eyes (410.56 ± 38.45μ m) and the right eyes of the control group (420.73±39.63μ m), and data of the contralateral eyes was lower than right eyes of the control group, distinct difference was shown between groups(P<0.05). The IMEM group's SFCT of sicked eyes and normal eyes at postoperative 1wk had no significant difference with that before operation (P>0.05), and at postoperative 1mo,SFCT of sicked eyes and normal eyes evidently increased, showing sharp difference compared with that before operation (P<0. 05). After that, the SFCT value stabilized, but there was no obvious difference between the sicked eyes and healthy eyes at postoperatively 1mo (P>0.05). The numbers of patients whose postoperative BCVA ≥ 0. 5 with different preoperative SFCT values had statistically significant differences (P<0.05), and those with BCVA ≥0.5 of SFCT values> 380μ m were significantly higher than those with <320μ m and 320μ m-380μ m groups. Fisher exact probability analysis showed that the differences were significant. Pearson analysis showed that there was a positive correlation between the postoperative choroid thickness and the best corrected visual acuity of IMEM group (r=0.629,P<0.05). · CONCLUSION: Choroidal thinning may be an important cause of IMEM, and preoperative choroidal thickness also has an influential effect on postoperative visual recovery.

Objective To observe the effect of stage-based acupuncture-moxibustion therapy on the endometrial thickness in patients suffering from repeated implantation failure in IVF-ET (in vitro fertilization and embryo transfer). Method Seventy-two patients suffering from repeated implantation failure in IVF-ET were randomized into two groups. Thirty-six cases in the treatment group were intervened by stage-based acupuncture-moxibustion therapy plus oral administration of Estradiol valerate tablets; the other 36 cases in the control group were prescribed with oral administration of Estradiol valerate tablets alone. The implantation result of IVF-ET was analyzed 3 cycles later. The endometrial thickness was compared before and after the intervention. Result The endometrial thickness of the non-pregnant women increased after the treatment in both groups (P<0.05), and the increase in the treatment group was more significant than that in the control group (P<0.05). The clinical pregnancy rate in the treatment group was significantly higher than that in the control group (P<0.05). Conclusion Stage-based acupuncture-moxibustion therapy can improve the endometrial thickness, promote the growth of endometrium, benefit the implantation of embryo, and enhance the clinical pregnancy rate.