The complete chloroplast genomes of Tetrastigma hemsleyanum (Vitaceae) from different regions of China: molecular structure, comparative analysis and development of DNA barcodes for its geographical origin discrimination.

BACKGROUND: Tetrastigma hemsleyanum is a valuable traditional Chinese medicinal plant widely distributed in the subtropical areas of China. It belongs to the Cayratieae tribe, family Vitaceae, and exhibited significant anti-tumor and anti-inflammatory activities. However, obvious differences were observed on the quality of T. hemsleyanum root from different regions, requiring the discrimination strategy for the geographical origins. RESULT: This study characterized five complete chloroplast (cp) genomes of T. hemsleynum samples from different regions, and conducted a comparative analysis with other representing species from family Vitaceae to reveal the structural variations, informative markers and phylogenetic relationships. The sequenced cp genomes of T. hemsleyanum exhibited a conserved quadripartite structure with full length ranging from 160,124 bp of Jiangxi Province to 160,618 bp of Zhejiang Province. We identified 112 unique genes (80 protein-coding, 28 tRNA and 4 rRNA genes) in the cp genomes of T. hemsleyanum with highly similar gene order, content and structure. The IR contraction/expansion events occurred on the junctions of ycf1, rps19 and rpl2 genes with different degrees, causing the differences of genome sizes in T. hemsleyanum and Vitaceae plants. The number of SSR markers discovered in T. hemsleyanum was 56-57, exhibiting multiple differences among the five geographic groups. Phylogenetic analysis based on conserved cp genome proteins strongly grouped the five T. hemsleyanum species into one clade, showing a sister relationship with T. planicaule. Comparative analysis of the cp genomes from T. hemsleyanum and Vitaceae revealed five highly variable spacers, including 4 intergenic regions and one protein-coding gene (ycf1). Furthermore, five mutational hotspots were observed among T. hemsleyanum cp genomes from different regions, providing data for designing DNA barcodes trnL and trnN. The combination of molecular markers of trnL and trnN clustered the T. hemsleyanum samples from different regions into four groups, thus successfully separating specimens of Sichuan and Zhejiang from other areas. CONCLUSION: Our study obtained the chloroplast genomes of T. hemsleyanum from different regions, and provided a potential molecular tracing tool for determining the geographical origins of T. hemsleyanum, as well as important insights into the molecular identification approach and and phylogeny in Tetrastigma genus and Vitaceae family.

Identifying the grade of bladder cancer cells using microfluidic chips based on impedance.

Quantification of tumor cell heterogeneity is critical for clinical diagnostic and therapeutic applications, including evaluation of the cancerous stage of tumors. In this work, we presented a novel method to effectively distinguish the grade of bladder cancer at a single-cell level in both cell line and clinical cell samples. This was achieved by taking advantage of microdroplets and microelectrodes, which can encapsulate and then trap single cells for measuring their impedance in a label-free and non-invasive manner. These findings suggested that this impedance analysis device based on droplet microfluidics is promising in the fields of clinical and point-of-care diagnostics.

Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships.

BACKGROUND: The Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species. RESULTS: The cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy. CONCLUSIONS: Overall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

The complete chloroplast genome sequence of Rubus hirsutus Thunb. and a comparative analysis within Rubus species.

Rubus hirsutus is a type of tonifying kidney-essence herb that belongs to the Rosaceae family, and has been commonly used to treat multiple diseases, such as polyuria, impotence, and infertility. In this study, we determined the complete chloroplast sequence of R. hirsutus and conduced a comparative analysis within the genus Rubus. The assembled chloroplast (cp.) genome is 156,380 bp in length with a GC content of 37.0% and shares a conserved quadripartite structure within the other cp. genomes in this genus. A total of 132 unique genes were annotated in the cp. genome of R. hirsutus, which contained 87 protein-coding genes, 37 tRNAs, and eight rRNAs. Seventeen duplicated genes were identified in the inverted repeats region. Furthermore, 70 simple sequence repeats and 35 long repeats were detected in total in the R. hirsutus chloroplast genome. Eight mutational hotspots were identified in the cp. genome of this species with higher nucleotide variations in non-coding regions than those of coding regions. Furthermore, the gene order, codon usage, and repeat sequence distribution were highly consistent in Rubus according to the results of a comparative analysis. A phylogenetic analysis indicated that there was a sister relationship between R. hirsutus and R. chingii. Overall, the complete chloroplast genome of R. hirsutus and the comparative analysis will help to further the evolutionary study, conservation, phylogenetic reconstruction, and development of molecular barcodes for the genus Rubus.

Cryptotanshinone enhances the efficacy of Bcr-Abl tyrosine kinase inhibitors via inhibiting STAT3 and eIF4E signalling pathways in chronic myeloid leukaemia.

CONTEXT: A portion of patients with chronic myeloid leukaemia (CML) develop resistance to the Bcr-Abl tyrosine kinase inhibitors (TKIs), limiting the clinical applications. Previous results have demonstrated the synergistic effects between cryptotanshinone (CPT) and imatinib on apoptosis of CML cells in vitro. OBJECTIVE: To determine the antileukemia effects of CPT and TKIs on the resistant CML cells, and further investigate the effect of combined treatment of CPT and imatinib on tumour growth and apoptosis in the xenograft model and clarify its regulatory mechanisms. MATERIALS AND METHODS: The combination effects of CPT and second-generation TKIs were evaluated in resistant CML cells K562-R. CPT and imatinib were orally administered once daily for 21 days on K562-R xenografts in nude mice (6 per group). Tumour proliferation and apoptosis were examined by Ki-67, PCNA and TUNEL staining. The expression levels of apoptotic markers and activities of STAT3 and eIF4E pathways were determined via immunohistochemistry staining and western blotting analysis. RESULTS: CPT significantly enhanced the antiproliferative effects of TKIs, via triggering cleavages of caspase proteins, and inhibiting activities of STAT3 and eIF4E pathways. The administration of CPT and imatinib dramatically inhibited the tumour growth of xenografts and achieved a suppression of 60.2%, which is 2.6-fold higher than that of single imatinib group. Furthermore, CPT and imatinib increased the apoptotic rates and markedly decreased the phosphorylation levels of STAT3 and eIF4E. CONCLUSIONS: Our results demonstrated that CPT could significantly enhance the antileukemia efficacy of TKIs, suggesting the therapeutic potential of CPT to overcome CML resistance.

[A review on cell-based models of human liver disease in vitro].

Unhealthy diet, habits and drug abuse cause a variety of liver diseases, including steatohepatitis, liver fibrosis, liver cirrhosis and liver cancer, which seriously affect human health. The fabrication of highly simulated cell models in vitro is important in the treatment of liver diseases and drug development. This article summarized the common strategies for the construction of liver pathology models in vitro. It introduced four typical cell models in vitro related to liver disease and provided a reference for the study of liver disease models.

[Current status and future prospect of pharmacotherapy for pancreatic cancer].

Pancreatic cancer is the most common digestive tract tumor with an increasing incidence in recent years. The poor prognosis of pancreatic cancer is mainly because of the inability of detecting tumor at an early stage,its high potential for early dissemination,and its relatively poor sensitivity to chemotherapy. Most patients have lost the opportunity for surgery when they are diagnosed,which resulted in an urgent need for the development of more effective and safe therapies for pancreatic cancer. However,the current clinical cancer chemotherapy based on gemcitabine leads to poor prognosis in pancreatic patients. With the continuous research on the biological and cellular signaling pathways of pancreatic cancer,there have emerged a great many of novel agents,including new chemotherapeutic,targetable and immune-modulatory drugs,and some drugs have achieved encouraging results. Furthermore,as an alternative and supplementary method,traditional Chinese medicine has shown good application prospects in the field of pancreatic cancer treatment. This article reviews the current status of drug therapy for pancreatic cancer,summarizes the strength and weakness of existing therapeutic drugs in the application process,gives prospects of possible breakthroughs for the pharmacotherapy in the future,and provides certain new ideas and lessons for subsequent drug development.

[Morphological characteristics identification and molecular DNA barcoding analysis of Hippocampus spinosissimus].

The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.

[Inhibitory effect and mechanism of platycodin D combined with imatinib on K562/R].

Platycodin D(PD) has a significantly inhibitory effect on multiple malignant tumors, and can inhibit the proliferation of leukemia cells K562 and induce apoptosis. However, its effect in improving the sensitivity of drug-resistant cells to imatinib and their molecular mechanism remained unclear. To investigate the effect and mechanism of PD alone or combined with imatinib (IM) in inhibiting CML imatinib resistant cell line K562/R, the cell proliferation was examined by CCK8 assay to reveal the effect of PD on the inhibitory function of imatinib. Cell apoptosis was detected by Annexin V-FITC/PI double staining. Protein expressions of cleaved caspase-3, cleaved caspase-9, PARP, cleaved PARP, Bcr/abl, p-AKT and p-mTOR were detected by Western blot. The results showed that the inhibitory effect of PD combined with imatinib on the proliferation and apoptosis of K562/R cells was significantly higher than that of the control group and the single drug group. Protein expressions of cleaved caspase-3, cleaved caspase-9 and cleaved PARP were significantly up-regulated in the combination group, and protein expressions of PARP, Bcr/abl, p-AKT and p-mTOR were down-regulated. The results indicated that PD increased the sensitivity of drug-resistant cells to imatinib, and the inhibitory effect of PD combined with imatinib was significantly better than the single drug on cell proliferation, induction of apoptosis, inhibition of Bcr/abl protein and PI3K/AKT/mTOR signaling pathway.

[Progress of regulation of leukemia stem cells of chronic myeloid leukemia by autophagy].

Leukemia stem cells (LSC) that were found in chronic myeloid leukemia (CML) responsible for the abnormal proliferation with the potential of self-renewal and multi-directional differentiation are involved in the pathophysiological process for drug resistance and relapse of CML. Autophagy, a conservative lysosomal degradation process that mediates cell degradation and recycling process, plays crucial roles in maintaining the homeostasis and function of intracellular environment. Recent studies suggested that autophagy is involved in the regulation of LSC differentiation and also closely related to the chemo-sensitivity of CML. In this review, we focused on the role of autophagy on chemotherapy sensitivity of CML as well as the leukemia stem cell function for the development of new anti-leukemia drugs.

Dihydrotanshinone induces apoptosis of SGC7901 and MGC803 cells via activation of JNK and p38 signalling pathways.

CONTEXT: Dihydrotanshinone (DHT), a natural compound from Salvia miltiorrhiza Bunge (Lamiaceae), showed higher cytotoxic potential compared with other tanshinones. Its effect and mechanism on gastric cancer have not been investigated. OBJECTIVE: This study evaluates the effects of DHT on cell proliferation and apoptosis on gastric cancer cells, and elucidates its molecular mechanisms. MATERIALS AND METHODS: Human gastric cancer MGC803 and SGC7901 cells were treated with various concentrations of DHT (0-15 µM) for 24 and 48 h, and cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell apoptosis was analysed by flow cytometry and DAPI staining. Western blots were performed to investigate changes in the level of apoptosis related genes in gastric cancer cell. RESULTS: DHT exhibited obvious inhibition of the survival of gastric cancer cells. The IC50 values in SGC7901 and MGC803 cells were 9.14 and 5.39 µM for 24 h, respectively. Cells treated with 6 µM DHT resulted in 41.3% and 35.4% apoptotic cell fractions in SGC7901 and MGC803 cells, respectively, significantly higher than that of the control. Hallmarks of apoptosis were observed in gastric cancer cells after DHT exposure. DHT enhanced the expression levels of cleaved caspase-3, caspase-9 and poly-ADP-ribose polymerases. Furthermore, DHT increased the phosphorylation of JNK and p38 in SGC7901 and MGC803 cells. CONCLUSION: DHT induced growth inhibition and apoptosis of gastric cancer cells, involving activation of caspase proteins and the JNK/p38 signaling pathway. The results indicated that DHT has a promising chemotherapeutic potential for human gastric cancer.

Facile Preparation of Bifunctional Monodisperse Nanospheres with Tunable Size and Luminescence.

Nanotechnology has found wide use in biomedical applications and the food and bioprocessing industry. In this light, we demonstrate a facile strategy to prepare bifunctional monodisperse silica nanospheres encapsulating chitosan-coated magnetic nanoparticles and CdTe quantum dots. The size of these composite spheres can be adjusted from 90 nm to 500 nm by varying the concentration of ammonia, water, tetraethyl orthosilicate, and the ratio of the chitosan-coated magnetic nanoparticles and CdTe quantum dots. The composite spheres are characterized using scanning electron microscope analyses, transmission electron microscope analyses, energy-dispersed spectrum studies, Malvern Zetasizer, vibrating sample magnetometer, and fluorescence microscopy. The spheres exhibit good monodispersion and favorable superparamagnetic and fluorescent properties. The luminescence of the spheres can be varied by using different types of coated quantum dots. Such composite spheres with tunable characteristics allow for external manipulation of research systems by magnetic fields together with the real-time fluorescent monitoring of multiple samples. The abovementioned properties can potentially be exploited for application in the biomedical and biosensing fields.

[Effect of cryptotanshinone on imatinib sensitivity and P-glycoprotein expression of chronic myeloid leukemia cells].

Cryptotanshinone (CPT), a lipid soluble active compound in Salvia miltiorrhiza, has a significant inhibitory effect on multiple malignant tumors, e. g. chronic myeloid leukemia (CML) cells and can effectively enhance imatinib's chemotherapeutic effect. However, its functional molecular mechanism remained unclear. In this experiment, the authors conducted a systematic study on the effect of CPT on the imatinib sensitivity and P-glycoprotein (P-gp) expression in CML cells by using CML cells K562 and imatinib persister K562-R. The MTT assays were performed to determine CPT's impact on the inhibitory effect of imatinib. Annexin V-FITC/PI staining analysis was used to detect the changes in the cell apoptosis rate. The active changes in apoptosis regulatory proteins Caspase-3, Caspase-9 and PARP were determined by Western blot. After the cells were pretreated with the gradient concentration of CPT, the expression of P-gp was analyzed by Western blot and flow cytometry. The changes in intracellular concentrations of imatinib were determined by HPLC analysis. The results indicated that the pretreatment with CPT significantly increased the proliferation inhibiting and apoptosis inducing effects of imatinib on K562 and K562-R cells as well as the degradation product expression of pro-apoptotic proteins Caspase-3, Caspase-9 and PARP, with a significant difference with the control group (P < 0.01). However, CPT showed no impact on the P-gp expression in CML cells and the intracellular concentrations of imatinib. In summary, the findings suggested that CPT enhanced the sensitivity of CML cells to imatinib. Its mechanism is not dependent on the inhibition in P-gp expression and the increase in intracellular drug concentration.

Rolling circle amplification immunoassay combined with gold nanoparticle aggregates for colorimetric detection of protein.

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[ε-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a "sandwich" structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.

The Tanshinones (Tan) Extract From Salvia miltiorrhiza Bunge Induces ROS-Dependent Apoptosis in Pancreatic Cancer via AKT Hyperactivation-Mediated FOXO3/SOD2 Signaling.

CONTEXT: Salvia miltiorrhiza (SM) is a commonly used herb in traditional Chinese medicine (TCM) and has been used in the treatment of pancreatic cancer to relieve the symptom of "blood stasis and toxin accumulation." Tanshinones (Tan), the main lipophilic constituents extracted from the roots and rhizomes of SM, have been reported to possess anticancer functions in several cancers. But the mechanism of how the active components work in pancreatic cancer still need to be clarified. OBJECTIVE: In this study, we aimed to investigate the therapeutic potential of Tan in pancreatic cancer and elucidate the underlying mechanisms. MATERIALS AND METHODS: The viabilities of PANC-1 and Bxpc-3 cells were determined by MTT assay, after treatment with various concentrations of Tan. The apoptotic cells were quantified by annexin V-FITC/PI staining and DAPI staining assays. The expression of relative proteins was used western blotting. Tumor growth was assessed by subcutaneously inoculating cells into C57BL/6 mice. RESULTS: Our experiments discovered that Tan effectively suppressed pancreatic cancer cell proliferation and promoted apoptosis. Mechanistically, we propose that Tan enhances intracellular ROS levels by activating the AKT/FOXO3/SOD2 signaling pathway, ultimately leading to apoptosis in pancreatic cancer cells. In vivo assay showed the antitumor effect of Tan. CONCLUSION: Tan, a natural compound from Salvia miltiorrhiza, was found to effectively suppress pancreatic cancer cell proliferation and promote apoptosis both in vitro and in vivo. Mechanistically, we propose a positive feedback loop mechanism. These findings provide valuable insights into the molecular pathways driving pancreatic cancer progression.

Capacitive Bionic Magnetic Sensors Based on One-Step Biointerface Preparation.

Magnetic biomolecule-based bionic magnetic field sensors are anticipated to open up novel pathways for magnetic field detection. The detection range and accuracy of current bionic magnetic field sensors are limited, and little work is based on the capacitive response principle. We successfully developed a biochemical interface with an extralarge target-receptor size ratio, which can be manufactured in a single step for weak magnetic field detection across a wide frequency range, and we used electrochemical capacitance as a magnetic field change conduction strategy. The thickness-controllable nanoscale bovine serum albumin/graphene layer on an indium tin oxide working electrode combines with the one-step preparation method to immobilize the MagR/Cry4 complex. This capacitive bionic magnetic sensor can achieve the detection range of 0-120 mT. This biointerface design strategy obtains the further improvement of the performance of this bionic magnetic field sensor. Furthermore, the biointerface construction and optimization methodology in this proposal has potential applications in the design of other medical biosensors.

Establishment of bladder cancer spheroids and cultured in microfluidic platform for predicting drug response.

Cisplatin-containing combination chemotherapy has been used as the standard treatment for bladder cancer patients at advanced stage. However, nearly 50% of patients are nonresponders. To guide the selection of more effective chemotherapeutic agents, a bladder cancer spheroids microfluidic drug sensitivity analysis system was established in this study. Bladder cancer spheroids were established and successfully cultured in a customized microfluidic device to assess their response to different chemotherapeutic agents. The in vitro drug sensitivity results were also compared to patient-derived xenograft (PDX) models and clinical responses of patients. As a result, bladder cancer spheroids faithfully recapitulate the histopathological and genetic features of their corresponding parental tumors. Furthermore, the in vitro drug sensitivity outcomes of spheroids (n = 8) demonstrated a high level of correlation with the PDX (n = 2) and clinical response in patients (n = 2). Our study highlights the potential of combining bladder cancer spheroids and microfluidic devices as an efficient and accurate platform for personalized selection of chemotherapeutic agents.

A colorimetric method for H1N1 DNA detection using rolling circle amplification.

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay has been successfully developed as a method for the effective detection of H1N1 DNA. Specific oligonucleotide and reporter primer probes were designed together with a circular template, and the oligonucleotide probes were attached to the surfaces of magnetic beads (MBs) to form functional MB-DNA conjugates as capture probes for the target H1N1 DNA molecules. Together with the addition of DNA targets and reporter primer probes to the MB-DNA conjugates, sandwiched hybrids were formed. The initiation of RCA amplification using the circular template in the presence of phi29 polymerase allowed for the amplification of a large number of repeat sequences of the single-stranded (ss)-DNA product. This RCA product accumulated gold nanoparticles (AuNPs), resulting in a colorimetric change that could be viewed by the naked eye or detected using UV-vis spectroscopy. According to this method, H1N1 DNA could be detected at the 1 pmol L(-1) level. This platform exhibited design convenience, simplicity, and cost-effectiveness, and could be used to provide a new diagnostic assay for H1N1, and other infectious diseases.

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101.

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.

Comparative chloroplast genome analysis of four Polygonatum species insights into DNA barcoding, evolution, and phylogeny.

The Polygonatum genus represents a perennial herb with the Liliaceae family, boasting substantial economic and medicinal significance. The majority of Polygonatum plants exhibit notable similarity while lacking distinctive identifying characteristics, thus resulting in the proliferation of adulterated medicinal materials within the market. Within this study, we conducted an in-depth analysis of the complete chloroplast (cp) genomes of four Polygonatum plants and compared them with four closely akin species. The primary objectives were to unveil structural variations, species divergence, and the phylogenetic interrelations among taxa. The cp genomes of the four Polygonatum species were typified by a conventional quadripartite structure, incorporating a large single copy region (LSC), a small single copy region (SSC), and a pair of inverted repeat regions. In total, we annotated a range of 131 to 133 genes, encompassing 84 to 86 protein-coding genes, 38 transfer RNA (tRNA) genes, 8 ribosomal RNA (rRNA) genes, and 0 to 2 pseudogenes (ycf1, infA). Our comparative analyses unequivocally revealed a remarkable consistency in gene order and GC content within the Polygonatum genus. Furthermore, we predicted a potential 59 to 64 RNA editing sites distributed across 22 protein-coding genes, with the ndhB gene exhibiting the most prominent propensity for RNA editing sites, boasting a tally of 15 sites. Notably, six regions of substantial potential variability were ascertained, characterized by elevated Pi values. Noteworthy, molecular markers for species identification, population genetic scrutiny, and phylogenetic investigations within the genus were identified in the form of the psaJ-rpl33 and trnS + trnT-psaD barcodes. The resultant phylogenetic tree unequivocally depicted the formation of a monophyletic clade comprising species within the evolutionary framework of Liliaceae, demonstrating closer evolutionary affinities with Maianthemum, Dracaeneae, and Asparageae. This comprehensive compendium of findings collectively contributes to the advancement of molecular species identification, elucidation of phylogenetic interrelationships, and the establishment of DNA barcodes tailored to the Polygonatum species.