Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.

Objective To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island. Methods A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned. Results There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1-mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1gene mutation site in 19 Pfubp1-mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene (Pfap2mu-inner1, Pfap2mu-inner2, Pfap2mu-inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu-mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu-mutant P. falciparum isolates (10.06%). Conclusions Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).

Objective: To explore the effect and mechanism of lycorine on oxaliplatin ( OXA) induced chemotherapy pain in mice． Methods : 40 mice were randomly divided into 4 groups，10 mice per group，which were respectively divided into control group，model group，administration group，and inhibitor group．A mouse model of chemotherapy induced pain was established by intraperitoneal injection of OXA for 5 consecutive days．Intrathecal administration of lycorine was performed．Behavioral changes and expression levels of inflammatory related proteins were detected . Results : Compared with control group，model group mice exhibited the increased number of spontaneous flinches，decreased mechanical nociceptive threshold，decreased movement distance and latency，and up-regulated expression levels of interleukin-1 β (IL-1 β) ，astrocytic marker glial fibrillary acidic protein ( GFAP) ，cyclooxygenase-2( COX-2) ，NOD-like receptor protein 3 ( NLRP3 ) ，cysteinyl aspartate and specific proteinase 1 ( Caspase- 1) ．Compared with model group，lycorine administration reduced the number of spontaneous flinches，increased mechanical nociceptive threshold ，enhanced the movement distance and latency ，bound and reduced COX-2 expression，down-regulated the expression levels of IL-1 β , GFAP ，NLRP3 and Caspase-1． Conclusion Lycorine reduces COX-2 expression，inhibits NLRP3 inflammasome activation，suppresses spinal inflammation，consequently alleviates pain behaviors and improved motor ability of mice.

Objective: To observe the root and root canal morphology of mandibular second molars in Western Guangxi by CBCT, to provide a reference for clinical diagnosis and treatment. Methods: In total, 564 patients′ 1 128 mandibular second molars that satisfy the inclusion criteria were analyzed with a planmecaromexis CBCT machine and its own image analysis software. The patients′ gender, age and ethnic differences in the root and canal morphology and the symmetry of the bilateral root and canal were statistically analyzed. Results: Among the 1 128 mandibular second molars, 662 were the Zhuang ethnic group and 384 were the Han ethnic group, and 82 were other ethnic groups; the double root type and C-shaped root type accounted for a relatively high proportion: 73.94% and 24.47%, respectively. The detection rates of the double root type were higher in males than in females (P < 0.05); the detection rates of the C-shaped root type were higher in females than in males (P <0.05); the root type of the teeth was mainly double-rooted in the Zhuang ethnic group (P<0.01). The incidence of type IV in the mesial root of the double root type mandibular second molar was the highest (P < 0.01), and the incidence of type I in the distal root was the highest (P < 0.01). The C-shaped root canal is more continuous at the mouth of the root canal, more downward corresponds to a worse continuity: in three different levels of root canal orifice, root middle and root apex, the root canal orifice is dominated by the C1 type, and both root middle and root apex are mainly C3-type (P < 0.01). The difference in symmetry of bilateral roots and root canals was statistically significant among different gender groups, age groups, and ethnic groups (P < 0.05): there were more males than females, the results in the 18-35-year-old group and the Zhuang ethnic group were higher. Conclusion The root and root canal morphology of mandibular second molars in western Guangxi people are complex and changeable. The roots are mainly double root type in the Han ethnic group and the Zhuang ethnic group. C-shaped roots are also common. The detection rate of C-shaped roots in the Zhuang ethnic group was higher, and the symmetry rate of bilateral roots and that of bilateral root canals was higher in the Zhuang ethnic group than in the Han ethnic group.

Objective To investigate the genetic polymorphisms of Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), chloroquine resistance transporter (PfCRT) and Kelch 13 (PfK13) genes in Bioko Island, Equatorial Guinea, so as to provide insights into the development of the malaria control strategy in local areas. Methods A total of 85 peripheral blood samples were collected from patients with Plasmodium falciparum infections in Bioko Island, Equatorial Guinea in 2018 and 2019, and genomic DNA was extracted. The PfMDR1, PfCRT and PfK13 genes were amplified using a nested PCR assay. The amplification products were sequenced, and the gene sequences were aligned. Results There were no mutations associated with artemisinin resistance in PfK13 gene in Bioko Island, Equatorial Guinea, while drug-resistant mutations were detected in PfMDR1 and PfCRT genes, and the proportions of PfMDR1_N86Y, PfMDR1_Y184F and PfCRT_K76T mutations were 35.29% (30/85), 72.94% (62/85) and 24.71% (21/85), respectively. Conclusion There are mutations in PfMDR1, PfCRT and PfK13 genes in P. falciparum isolates from Bioko Island, Equatorial Guinea.

To investigate the potential molecular mechanism of the combination of Platycodonis Radix and Lilii Bulbus with the homology of medicine and food in the treatment of pneumonia by means of network pharmacology and in vitro verification experiment. Under the condition of bioavailability(OB)≥30% and drug-like(DL)≥0.18, the active components of Platycodonis Radix and Lilii Bulbus were screened in TCMSP database; the prediction targets of active components were searched from TCMSP, DrugBank and other databases, and the potential targets of pneumonia were obtained through GeneCards and OMIM database. The common targets were obtained by the intersection of drug and disease targets. The PPI network of common targets was constructed by STRING 11.0, and the core targets were obtained by topological analysis. Then the core targets received GO and KEGG analysis with use of WebGestalt and Metascape. The "component-target-pathway" network was constructed with the help of Cytoscape 3.7.1 software, and the component-target molecular docking verification was carried out with Discovery Studio 2016 software. Finally, the core targets and pathways were preliminarily verified in vitro. In this study, 12 active components were screened, 225 drug prediction targets and 420 potential diseases targets were obtained based on data mining method, and 14 core targets were obtained by topological analysis, including TNF, MMP9, AKT1, IL4 and IL2. The enrichment results of GO and KEGG showed that "Platycodonis Radix and Lilii Bulbus" drug pair may regulate inflammation, cell growth and metabolism by acting on 20 key signaling pathways such as TNF and IL-17, thereby exerting anti-pneumonia effects. The results of molecular docking showed that 12 active components had good binding ability with 14 core targets. In vitro experiment results showed that the core components of "Platycodonis Radix and Lilii Bulbus" drug pair could inhibit the expression of MMP9 and TNF-α by regulating TNF signal pathway. This study confirmed the scientificity and reliability of the prediction results of network pharmacology, and preliminarily revealed the potential molecular mechanism of the compatibility of Platycodonis Radix and Lilii Bulbus in the treatment of pneumonia. It provides a novel insight on systematically exploring the mechanism of the compatible use of Platycodonis Radix and Lilii Bulbus, and has a certain reference value for the research, development and application of new drugs.
Drugs, Chinese Herbal ; Humans ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Pneumonia/drug therapy* ; Reproducibility of Results

This article introduced the main biological mechanisms of acupuncture promoting nerve function recovery after spinal cord injury, which include inhibition of inflammation and oxidative stress, alleviation of neuropathic pain, increase of neurotrophic active sub-stance, regulation of cell survival/apoptosis gene and neural regeneration pathway.

Objective To investigate the effect of Santong electroacupuncture (EA) on mRNA and protein expression of p75 neurotroph-in receptor (p75NTR) in rats with spinal cord injury (SCI). Methods A total of 72 female Sprague-Dawley rats were randomly assigned to sham operation group (group A, n=8) and model group (n=64). In the model group, Allen's method was used to make SCI rats model, in which 48 survived model rats were further subdivided into model control group (group B, n=12), EA group (group C, n=12), inhibitor Nogo extra cellular peptide residues 1-40 (NEP1-40) group (group D, n=12) and EA+inhibitor NEP1-40 group (group E, n=12) according to de-sign proposal. The treatment groups were electroacupunctured on Dazhui (GV14) and Yaoyangguan (GV3), bilateral Ciliao (BL32) and Zu-sanli (ST36) with loose-tight wave, for 20 minutes every day. After 7 and 14 days of treatment, injured spinal cord tissue was extracted for detecting. The mRNA and protein expression of p75NTR was detected by real-time fluorescent quantitative PCR, in situ hybridization and Western blotting respectively. The hind limb motor function was assessed with Basso-Beattie-Bresnahan (BBB) score. Results The BBB score increased in the treatment groups compared with group B, and was higher in group E than in groups C and D (P<0.05), as well as on the 14th day than on the 7th day in all the treatment groups (t>2.623, P<0.05). The mRNA and protein expression of p75NTR in spinal cord tissues decreased in the treatment groups compared with group B (P<0.05), and no significant difference was found among the treatment groups (P>0.05). Conclusion Santong elerctroacupuncture treatment could improve the hind limb motor function, which may associate with inhibition of the mRNA and protein expression of p75NTR in rats after SCI.

Objective To investigate the effect of suspended moxa stick moxibustion on points Shenshu(BL23) and Guanyuan(CV4) on the pituitary-adrenal axis and the pituitary-thyroid axis in rats with kidney yang deficiency. Method A rat model of corticosterone kidney yang deficiency was made by intramuscular injection of hydrocortisone. The rats were randomized into model control and moxibustion treatment groups. A blank control group was set up. The moxibustion treatment group received suspended moxa stick moxibustion on points Shenshu and Guanyuan 20 min once daily, for a total of 14 times. After the completion of treatment, serum CORT, ACTH, T3, T4 and TSH contents were measured by ELISA and pituitary expressions of ACTH and TSH mRNA were determined by RT-PCR. Result There was no significant difference in serum CORT (P>0.05), there were significant differences in serum ACTH, T4 and TSH and pituitary ACTH and TSH mRNA (P<0.05) and there was a very significant difference in serum T3 (P<0.01) between the moxibustion treatment and model control groups. There were no significant differences in the above indicators between the moxibustion treatment and blank control groups (P>0.05). Conclusion Suspended moxa stick moxibustion on points Shenshu and Guanyuan produces a therapeutic effect on rat kidney yang deficiency by decreasing serum TSH content, down-regulating pituitary TSH mRNA expression, increasing serum ACTH, T3 and T4 contents and up-regulating pituitary ACTH mRNA expression.

Objective To observe the effect of acupuncture on the expressions of serum corticotropin-releasing hormone (CRH), pituitary adrenocorticotropic hormone (ACTH) and its mRNA, to discuss the predominant and secondary factors that influence the efficacy of acupuncture, and to explore the optimal combination of acupuncture parameters.Method The rat model of kidney yang deficiency was made by muscular injection with hydrocortisone. Serum CRH, pituitary ACTH and its mRNA expressions were taken as the observation indexes, and 4 influential factors (i.e. selection of acupoints, angle of needle twisting, frequency of needle twisting, and time of needle twisting) plus 3 levels of each factor were arranged to L9(34) orthogonal table. A computer-controlled acupuncture manipulation device was used to treat the kidney yang rats. Serum CRH and pituitary ACTH were detected by enzyme-linked immunosorbent assay (ELISA) and pituitary ACTH mRNA expression by real time fluorescent quantitative PCR, to illustrate the primary and secondary factors that affect the acupuncture effectiveness.Results Regarding the effect on acupuncture effectiveness, the time of needle twisting ranked the top, followed by frequency of needle twisting, angle of needle twisting, and selection of acupoints. Angle of needle twisting, frequency of needle twisting, and time of needle twisting should be the predominant factors or secondary factors in influencing acupuncture effectiveness (P<0.01 orP>0.05). Selection of acupoints should be an important factor or secondary factor in influencing acupuncture effectiveness (P<0.05 orP>0.05).Conclusion For rats with kidney yang deficiency, the optimal acupuncture parameters combination should be acupuncture at Shenshu (BL 23), with twisting angle by 90°, twisting frequency at 180/min, and needle twisting time 120 s. Different factors can produce different effects on the therapeutic efficacy of acupuncture in treating rats with kidney yang deficiency; among the factors, the time of needle twisting produces the most significant effect, followed by frequency of needle twisting, angle of needle twisting, and selection of acupoints.