Tendon stem/progenitor cells are promising reparative cell sources for multiple musculoskeletal injuries of concomitant articular cartilage lesions associated with ligament injuries.

BACKGROUND: Trauma-related articular cartilage lesions usually occur in conjunction with ligament injuries. Torn ligaments are frequently reconstructed with tendon autograft and has been proven to achieve satisfactory clinical outcomes. However, treatments for the concomitant articular cartilage lesions are still very insufficient. The current study was aimed to evaluate whether stem cells derived from tendon tissue can be considered as an alternative reparative cell source for cartilage repair. METHODS: Primary human tendon stem/progenitor cells (hTSPCs) were isolated from 4 male patients (32 ± 8 years) who underwent ACL reconstruction surgery with autologous semitendinosus and gracilis tendons. The excessive tendon tissue after graft preparation was processed for primary cell isolation with an enzyme digestion protocol. Decellularization cartilage matrix (DCM) was used to provide a chondrogenic microenvironment for hTSPCs. Cell viability, cell morphology on the DCM, as well as their chondrogenic differentiation were evaluated. RESULTS: DAPI staining and DNA quantitative analysis (61.47 µg per mg dry weight before and 2.64 µg/mg after decellularization) showed that most of the cells in the cartilage lacuna were removed after decellularization process. Whilst, the basic structure of the cartilage tissue was preserved and the main ECM components, collagen type II and sGAG were retained after decellularization, which were revealed by DMMB assay and histology. Live/dead staining and proliferative assay demonstrated that DCM supported attachment, survival and proliferation of hTSPCs with an excellent biocompatibility. Furthermore, gene expression analysis indicated that chondrogenic differentiation of hTSPC was induced by the DCM microenvironment, with upregulation of chondrogenesis-related marker genes, COL 2 and SOX9, without the use of exogenous growth factors. CONCLUSION: DCM supported hTSPCs attachment and proliferation with high biocompatibility. Moreover, TSPCs underwent a distinct chondrogenesis after the induction of a chondrogenic microenvironment provided by DCM. These results indicated that TSPCs are promising reparative cell sources for promoting cartilage repair. Particularly, in the cohort that articular cartilage lesions occur in conjunction with ligament injuries, autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction. In future clinical practice, combined ligament reconstruction with TSPCs- based therapy for articular cartilage repair can to be considered to achieve superior repair of these associated injuries, in which autologous TSPCs can be isolated from a portion of the tendon autograph harvested for ligaments reconstruction.

The role of IL-6/JAK/STAT signal in female infertility caused by hydrosalpinx.

INTRODUCTION: To explore the role of IL-6/JAK/STAT signaling in tubal infertility. METHODS: The fimbriae tissues of 14 patients with a history of infertility and hydrosalpinx and 14 patients with no history of infertility and no fallopian tube disease were collected. The tissues were then divided into hydrosalpinx group and control group followed by analysis of the protein expression of key factors in the IL-6/JAK/STAT signaling by immunohistochemistry and Western blot. RESULTS: Immunohistochemical staining showed significantly higher level of IL-6, JAK1, p-JAK1, JAK2, p-JAK2, STAT1, p-STAT1, STAT3, and p-STAT3 in hydrosalpinx group than those in control group with IL-6 being mainly located in the cytoplasm and p-JAK2, STAT1, p-STAT1, STAT3, and p-STAT3 in the cytoplasm and nucleus. JAK1 and p-JAK1 was mainly located in the cytoplasm and JAK2 is in the cytoplasm and nucleus without difference of their expression between two groups. Consistently, hydrosalpinx group presented significantly higher protein levels of IL-6, JAK1, p-JAK1, JAK2, p-JAK2, STAT1, p-STAT1, STAT3, and p-STAT3 than control group without difference of JAK1, p-JAK1, JAK2 level. CONCLUSION: The activation of IL-6/JAK2/STAT1 and STAT3 signaling pathways are found in the hydrosalpinx in infertile patients, indicating that they might be involved in the pathogenesis of hydrosalpinx.

Clinical analysis of high-intensity focused ultrasound (HIFU) combined with hysteroscopy-guided suction curettage (HGSC) in patients with cervical pregnancy.

OBJECTIVE: To evaluate the effectiveness of high-intensity focused ultrasound (HIFU) combined with hysteroscopy-guided suction curettage (HGSC) in treating cervical pregnancy. MATERIALS AND METHODS: This is a retrospective study. Seven patients with cervical pregnancy who visited the Third Xiangya Hospital of Central South University from January 2015 to December 2020 were enrolled in the current study. All seven patients were treated with HIFU under conscious sedation. All of them underwent HGSC at an average of 2 ± 1 days (range: 1-3 days) after HIFU. Before the therapy, the patient's clinical characteristics were collected, including duration of amenorrhea, gravidity and parity, the patient history of cesarean section and miscarriage, and the size of the gestational sac. The levels of ß-hCG and hemoglobin in serum were also reviewed. To assess the clinical outcomes of this combined treatment, the suction time of HGSC, bleeding volume, the clearance time of ß-hCG, and the time with returning of menstruation were evaluated. RESULTS: All seven patients (average age: 31 ± 6 years) have experienced amenorrhea (duration range, 48 ± 8 days) before the treatment of HIFU. The average number of pregnancies was four, and the number of deliveries was one. Previous medical history showed six patients had cesarean sections, and five patients have been miscarriages. After HIFU treatment, the fetal heartbeats were stopped in all seven patients based on the diagnosis by doppler ultrasound. The bleeding of gestational tissue decreased significantly. All patients had only mild lower abdominal pain, no fever, intestinal damage, or other complications were reported. The average operation time of operative suction curettage was 21 ± 9 min (range: 9-32 min), and the median bleeding volume was 10 ± 8 mL (range: 2-20 mL). Follow-up observations showed that the menstruations were returned in patients at an average of 38 ± 9 days (range: 30-50 days) after the treatment. The ß-hCG decreased from 41773 ± 32242 mIU/mL to 13101 ± 8454 mIU/mL in 29 ± 10 days after surgery. CONCLUSION: Based on these results with small subjects, we concluded that HIFU combined with HGSC might be an effective and safe treatment for patients with cervical pregnancy.

Three-dimensional gold nanowires with high specific surface area for simultaneous detection of heavy metal ions.

Traditional detection methods to detect heavy metal ions are time-consuming, complicated, and expensive. Here, we developed a simple electroless plating method to prepare three-dimensional gold nanowire (Au NW) films with high specific surface area. In an aqueous plating bath, tetrachloroauric acid, 4-dimethylaminopyridine and formaldehyde are used as precursor, ligand, and reducing agent, respectively. An electrochemical sensor based on a Au NWs/SPE could be applied for simultaneous detection of lead (Pb(II)), arsenic (As(III)), and mercury (Hg(II)) ions. The detection limits of Pb(II), As(III), and Hg(II) are 2.6, 1.5, and 4.2 µg L-1, all lower than the permissible limits of the WHO for drinking water (the permissible level of Pb(II) and As(III) is 10.0 µg L-1, and the permissible level of Hg(II) is 6.0 µg L-1), respectively. This work presents a simple and novel method to prepare gold nanowires for quick detection of trace heavy metal ions.

Food Traceability Systems, Consumers' Risk Perception, and Purchase Intention: Evidence from the "4-label-1-Q" Approach in Taiwan.

ABSTRACT: Many food safety issues have arisen in Taiwan during the past decade. Therefore, in 2016, the Taiwan government proposed the "five rings of food safety" policy to comprehensively protect consumer food supply. Among these policies, the "4-labels-1-Q" approach was adopted to ensure the selection of foods with traceable labels for retrospective study. Hence, this study investigated the association between the degree of familiarity with the 4-labels-1-Q food traceability system and risk perceptions and also investigated whether a consumer's purchase intention toward fresh foods with food labels changed after viewing an educational film on food labels. This study defined subjects as the main food purchasers for their families; 290 valid questionnaire interviews were administered and educational films shown in Tainan markets and stores. Results showed that knowledge about labels significantly affected risk perception for labeling. Age, educational level, and degree of risk perception influenced purchase intention. Results also showed that after viewing the video, subjects' label knowledge and purchase intention increased significantly. However, after adjustment for age, educational level, income, and purchase places, the effect of film education on risk perception was insignificant, especially for those who had lower educational levels, including those older than 65 years. Public trust can be boosted through label education among age groups using different channels and methods, and encouraging the sale of labeled foods in traditional markets would be a useful strategy. Age, educational level, income, and risk perception of participants significantly affected purchase intention. This study can be a reference for designing risk communication strategies and promoting traceable agricultural products.

Genome-Wide Analysis of Genetic Diversity in Plasmodium falciparum Isolates From China-Myanmar Border.

Plasmodium falciparum isolates from China-Myanmar border (CMB) have experienced regional special selective pressures and adaptive evolution. However, the genomes of P. falciparum isolates from this region to date are poorly characterized. Herein, we performed whole-genome sequencing of 34 P. falciparum isolates from CMB and a series of genome-wide sequence analyses to reveal their genetic diversity, population structures, and comparisons with the isolates from other epidemic regions (Thai-Cambodia border, Thai-Myanmar border, and West Africa). Totally 59,720 high-quality single-nucleotide polymorphisms (SNPs) were identified in the P. falciparum isolates from CMB, with average nucleotide diversity (π = 4.59 × 10-4) and LD decay (132 bp). The Tajima's D and Fu and Li's D values of the CMB isolates were -0.8 (p < 0.05) and -0.84 (p < 0.05), respectively, suggesting a demographic history of recent population expansion or purifying selection. Moreover, 78 genes of the parasite were identified that could be under positive selection, including those genes conferring drug resistance such as pfubp1. In addition, 33 SNPs were identified for tracing the source of the parasites with a high accuracy by analysis of the most differential SNPs among the four epidemic regions. Collectively, our data demonstrated high diversity of the CMB isolates' genomes forming a distinct population, and the identification of 33-SNP barcode provides a valuable surveillance of parasite migration among the regions.

Introduction of F446I mutation in the K13 propeller gene leads to increased ring survival rates in Plasmodium falciparum isolates.

BACKGROUND: Mutations in the Plasmodium falciparum k13 gene are associated with artemisinin (ART) resistance. However, it is unclear whether the F446I mutation, the most prevalent allele at the China-Myanmar border and north of Myanmar, is associated with ART resistance. Therefore, the aim of this study was to investigate the role of this mutation in ART resistance by generating transgenic parasites expressing the F446I mutant allele. METHODS: The transgenic parasites carrying the F446I or C580Y mutation in both 3D7 and FCC1/HN isolates were generated by single crossing-over recombination and verified using PCR and gene sequencing. The ring-stage survival assay of 0-3 h (RSA0-3 h) was used to evaluate ART susceptibility of the transgenic parasites in vitro. RESULTS: Four transgenic parasite lines named 3D7F446I mut, 3D7C580Y mut, FCC1/HNF446I mut and FCC1/HNC580Y mut were successfully generated. These parasite lines showed no changes in the expression level of k13 when compared with their parent parasite isolates. However, introduction of the F446I mutation in k13 of the 3D7 and FCC1/HN isolates led to elevated ring survival rates detected using RSA0-3 h when subjected to both 700 and 20 nM concentrations of dihydroartemisinin. The survival rates were similar to those detected in the parasite lines with the C580Y mutation. CONCLUSIONS: Insertion of the F446I mutation in k13 led to increased ring survival, suggesting that this mutation may be associated with ART resistance and could be used as a molecular marker for monitoring ART-resistant parasites. The results also highlights the importance of surveillance of F446I mutants for containing the resistant parasite.

Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border.

The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0-3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced.

Distinctive origin and spread route of pyrimethamine-resistant Plasmodium falciparum in southern China.

Southeast Asia (the Thailand-Cambodia border) has been considered the primal epicenter for most antimalarial drug resistance; however, numerous molecular epidemiological studies have successively reported multiple independent origins of sulfadoxine-pyrimethamine (SP) resistance-associated Plasmodium falciparum dhfr (pfdhfr) and pfdhps alleles in other areas. To better understand the origin and evolutionary pathway of the SP resistance in Southeast Asia, a total of 374 P. falciparum field isolates from the Yunnan-Burma border and Hainan Island in southern China have been collected for comprehensive investigations on the mutation patterns of the pfdhfr/pfdhps genes as well as their microsatellite haplotypes. By comparative analysis of single-nucleotide polymorphism (SNP) genotyping and flanking microsatellite haplotypes, we reveal a unique origin of pyrimethamine-resistant mutations in Pfdhfr gene in Hainan Island and an oriented spread route of the pyrimethamine resistance from the Thailand-Cambodia border into the Hainan area, which reflects the geographical traits and SP administration histories in the two geographically independent areas. Moreover, genetic linkages between the high-level SP resistance-conferring pfdhfr/pfdhps alleles have been established in the isolates from the Yunnan-Burma border, raising the concern of a genetic basis in adopting combination chemotherapies against falciparum malaria.

A critical role of perinuclear filamentous actin in spatial repositioning and mutually exclusive expression of virulence genes in malaria parasites.

Many microbial pathogens, including the malaria parasite Plasmodium falciparum, vary surface protein expression to evade host immune responses. P. falciparium antigenic variation is linked to var gene family-encoded clonally variant surface protein expression. Mututally exclusive var gene expression is partially controlled by spatial positioning; silent genes are retained at distinct perinuclear sites and relocated to transcriptionally active locations for monoallelic expression. We show that var introns can control this process and that var intron addition relocalizes episomes from a random to a perinuclear position. This var intron-regulated nuclear tethering and repositioning is linked to an 18 bp nuclear protein-binding element that recruits an actin protein complex. Pharmacologically induced F-actin formation, which is restricted to the nuclear periphery, repositions intron-carrying episomes and var genes and disrupts mutually exclusive var gene expression. Thus, actin polymerization relocates var genes from a repressive to an active perinuclear compartment, which is crucial for P. falciparium phenotypic variation and pathogenesis.

From in vivo to in vitro: dynamic analysis of Plasmodium falciparum var gene expression patterns of patient isolates during adaptation to culture.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages.

Identification and characterization of novel microRNAs from Schistosoma japonicum.

BACKGROUND: Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs. METHODOLOGY AND RESULTS: We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18-26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host. CONCLUSIONS: Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.

No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection.

BACKGROUND: The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs. RESULTS: Of 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum. METHODS: Short RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO) of miR-451. CONCLUSION: These results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a component to maintain the normal function of mature RBCs.