COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Analysis of the phytochemical components of Prunella vulgaris using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with molecular networking and assessment of their antioxidant and anti-α-glucosidase activities.

Prunella vulgaris has long been used in traditional medicine and is consumed as a tea in China. Here, the total phenolic and flavonoid concentrations of plants from different geographical regions were measured. It was found that the total phenolic acid concentration ranged from 4.15 to 8.82 g of gallic acid equivalent per 100 g of dry weight (DW), and the total flavonoid concentration was 4.67-7.33 g of rutin equivalent per 100 g DW. Antioxidant activities were measured using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and the results ranged from 73.47% to 94.43% and 74.54% to 93.39%, respectively, whereas α-glucosidase inhibition was between 75.31% and 95.49%. Correlation analysis showed that the total flavonoids in P. vulgaris had superior antioxidant and anti-α-glucosidase activities compared to the total phenolic compounds. The active components of P. vulgaris were analyzed using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry combined with both classical molecular networking and feature-based molecular networking on the Global Natural Products Social platform, identifying 32 compounds, namely 14 flavonoids, 12 phenolic compounds, and 6 other chemical components. These results could provide useful information on the use of P. vulgaris as a functional tea.

Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis.

BACKGROUND: The coronavirus disease 2019 (COVID-19) outbreak is evolving rapidly worldwide. OBJECTIVE: To evaluate the risk of serious adverse outcomes in patients with COVID-19 by stratifying the comorbidity status. METHODS: We analysed data from 1590 laboratory confirmed hospitalised patients from 575 hospitals in 31 provinces/autonomous regions/provincial municipalities across mainland China between 11 December 2019 and 31 January 2020. We analysed the composite end-points, which consisted of admission to an intensive care unit, invasive ventilation or death. The risk of reaching the composite end-points was compared according to the presence and number of comorbidities. RESULTS: The mean age was 48.9 years and 686 (42.7%) patients were female. Severe cases accounted for 16.0% of the study population. 131 (8.2%) patients reached the composite end-points. 399 (25.1%) reported having at least one comorbidity. The most prevalent comorbidity was hypertension (16.9%), followed by diabetes (8.2%). 130 (8.2%) patients reported having two or more comorbidities. After adjusting for age and smoking status, COPD (HR (95% CI) 2.681 (1.424-5.048)), diabetes (1.59 (1.03-2.45)), hypertension (1.58 (1.07-2.32)) and malignancy (3.50 (1.60-7.64)) were risk factors of reaching the composite end-points. The hazard ratio (95% CI) was 1.79 (1.16-2.77) among patients with at least one comorbidity and 2.59 (1.61-4.17) among patients with two or more comorbidities. CONCLUSION: Among laboratory confirmed cases of COVID-19, patients with any comorbidity yielded poorer clinical outcomes than those without. A greater number of comorbidities also correlated with poorer clinical outcomes.

Clinical characteristics and outcomes of hospitalised patients with COVID-19 treated in Hubei (epicentre) and outside Hubei (non-epicentre): a nationwide analysis of China.

BACKGROUND: During the outbreak of coronavirus disease 2019 (COVID-19), consistent and considerable differences in disease severity and mortality rate of patients treated in Hubei province compared to those in other parts of China have been observed. We sought to compare the clinical characteristics and outcomes of patients being treated inside and outside Hubei province, and explore the factors underlying these differences. METHODS: Collaborating with the National Health Commission, we established a retrospective cohort to study hospitalised COVID-19 cases in China. Clinical characteristics, the rate of severe events and deaths, and the time to critical illness (invasive ventilation or intensive care unit admission or death) were compared between patients within and outside Hubei. The impact of Wuhan-related exposure (a presumed key factor that drove the severe situation in Hubei, as Wuhan is the epicentre as well the administrative centre of Hubei province) and the duration between symptom onset and admission on prognosis were also determined. RESULTS: At the data cut-off (31 January 2020), 1590 cases from 575 hospitals in 31 provincial administrative regions were collected (core cohort). The overall rate of severe cases and mortality was 16.0% and 3.2%, respectively. Patients in Hubei (predominantly with Wuhan-related exposure, 597 (92.3%) out of 647) were older (mean age 49.7 versus 44.9 years), had more cases with comorbidity (32.9% versus 19.7%), higher symptomatic burden, abnormal radiologic manifestations and, especially, a longer waiting time between symptom onset and admission (5.7 versus 4.5 days) compared with patients outside Hubei. Patients in Hubei (severe event rate 23.0% versus 11.1%, death rate 7.3% versus 0.3%, HR (95% CI) for critical illness 1.59 (1.05-2.41)) have a poorer prognosis compared with patients outside Hubei after adjusting for age and comorbidity. However, among patients outside Hubei, the duration from symptom onset to hospitalisation (mean 4.4 versus 4.7 days) and prognosis (HR (95%) 0.84 (0.40-1.80)) were similar between patients with or without Wuhan-related exposure. In the overall population, the waiting time, but neither treated in Hubei nor Wuhan-related exposure, remained an independent prognostic factor (HR (95%) 1.05 (1.01-1.08)). CONCLUSION: There were more severe cases and poorer outcomes for COVID-19 patients treated in Hubei, which might be attributed to the prolonged duration of symptom onset to hospitalisation in the epicentre. Future studies to determine the reason for delaying hospitalisation are warranted.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Clinical significance of common-stem lenticulostriate arteries in patients with internal watershed infarction.

BACKGROUND: A common-stem origin of lenticulostriate arteries (CS-LSAs) is an anatomical variation that supplies a moderate to large section of the basal ganglia. We hypothesized that CS-LSAs with a patent orifice are located at distal positions of the acute-occluded middle cerebral artery (MCA) and that the blood flow of CS-LSAs is supplied by pail arterial anastomoses and results in hypoperfusion of CS-LSAs, similar to a deep watershed (DWS) infarction. OBJECTIVE: Our study evaluated the possibility of CS-LSAs in patients with DWS infarction and MCA occlusion and also assessed the safety of endovascular therapy (ET) in these patients. METHODS: A cohort of consecutive patients with DWS infarction and MCA occlusion and in whom full recanalization via ET was achieved were identified. Patients were divided into two groups based on the presence of CS-LSAs observed during ET. In addition, radiological and clinical data were retrospectively analyzed. RESULTS: Thirty-three patients were included, and CS-LSAs were observed in 48.5% (16/33) of patients. The possibility (72.2%, 13/18) of CS-LSAs was high in patients with DWS infarction companied with basal ganglia infarction. A good clinical outcome was similar in patients with CS-LSAs and basal ganglia infarction and in patients without CS-LSAs and basal ganglia infarction (69.2% vs. 81.8%, P = 0.649). CONCLUSIONS: The possibility of CS-LSAs was 48.5% in patients with DWS infarction and MCA occlusion, and the revascularization procedure was safe and feasible in these patients despite the moderate-to-large basal ganglia infarction.

Perineal Assessment Tool (PAT-C): Validation of a Chinese Language Version and Identification of a Clinically Validated Cut Point Using ROC Curve Analysis.

PURPOSE: The purpose of this study was to evaluate content validity and feasibility of the Chinese language perineal assessment tool (PAT-C), to assess its use in the clinical setting, and establish an optimal cut point for identifying patients at high risk for incontinence-associated dermatitis (IAD). DESIGN: Psychometric evaluation of existing instrument. SUBJECTS AND SETTING: The sample comprised 440 patients managed in intensive care units of 3 hospitals across the island of Taiwan and an affiliated home care service. One hundred three nurses practicing throughout Taiwan participated in the workshops in the third phase of the experiment. METHODS: The content validity of the PAT-C was evaluated by 3 experienced nurses using the content validity index statistic. We calculated a receiver operating characteristics (ROC) curve to determine a cut point of high-risk IAD. The curve was based on assessment of patients from receiving care from the intensive care unit and home care service of Cathay General Hospital (located in Taipei, New Paipei and Hsinchu). Nurse perceptions on the feasibility of PAT-C were assessed using an investigator-developed survey. RESULTS: Three experienced nurses rated the PAT-C and gave a robust overall content validity index score of 97.22%. The cut point for identifying patients at high risk for developing IAD via ROC curve analysis of 440 patients was 7.5 (sensitivity: 0.85; specificity: 0.79, area under curve: 0.82, P value < .001). One hundred three enrolled nurses attended the workshops and evaluated the feasibility using the PAT-C. Most of the participants considered the PAT-C as necessary (97.90%), 49.7% of participants suggested IAD risk assessment should be implemented by first-line (generalist) nurses, and 40% of participants recommended assessment on a daily basis. CONCLUSIONS: Study findings indicate robust content validity, and results of the investigator survey of nurse perceptions of the PAT-C indicate the potential for its widespread use in the clinical setting. We found that a cut point score 8 or more indicates a high risk for developing IAD.

Long-term efficacy of a hepatitis E vaccine.

BACKGROUND: Hepatitis E virus (HEV) is a leading cause of acute hepatitis. The long-term efficacy of a hepatitis E vaccine needs to be determined. METHODS: In an initial efficacy study, we randomly assigned healthy adults 16 to 65 years of age to receive three doses of either a hepatitis E vaccine (vaccine group; 56,302 participants) or a hepatitis B vaccine (control group; 56,302 participants). The vaccines were administered at 0, 1, and 6 months, and the participants were followed for 19 months. In this extended follow-up study, the treatment assignments of all participants remained double-blinded, and follow-up assessments of efficacy, immunogenicity, and safety were continued for up to 4.5 years. RESULTS: During the 4.5-year study period, 60 cases of hepatitis E were identified; 7 cases were confirmed in the vaccine group (0.3 cases per 10,000 person-years), and 53 cases in the control group (2.1 cases per 10,000 person-years), representing a vaccine efficacy of 86.8% (95% confidence interval, 71 to 94) in the modified intention-to-treat analysis, rather than (95% confidence interval, 71 to 84) [corrected]. Of the participants who were assessed for immunogenicity and were seronegative at baseline, 87% of those who received three doses of the hepatitis E vaccine maintained antibodies against HEV for at least 4.5 years; HEV antibody titers developed in 9% in the control group. The rate of adverse events was similar in the two groups. CONCLUSIONS: Immunization with this hepatitis E vaccine induced antibodies against HEV and provided protection against hepatitis E for up to 4.5 years. (Funded by the Chinese Ministry of Science and Technology and others; ClinicalTrials.gov number, NCT01014845.).

[Isolation and expression analysis of a hexose transporter gene DoHT1 in Dendrobium officinale].

In this study, RACE technology was employed to isolate the full length cDNA of DoHT1 in Dendrobium officinale, followed by bioinformatics analysis of the sequence characteristics. And the expression pattern of the gene was also analyzed by quantitative PCR. The full length cDNA of DoHT1 was 1 586 bp in length, containing a 1 536 bp ORF, which encoded a 511-aa protein with molecular weight of 56.18 kD and isoelectric point of 9.08. The deduced DoHT1 protein had the major facilitator superfamily conserved domain (22-483), SUGAR₋TRANSPORT₋1 (139-164), and SUGAR₋TRANSPORT₋2 (338-355), typical for sugar transporter; DoHT1, without a single peptide had 11 transmembrane regions, and was predicted to locate in the plasma membrane; DoHT1 had high identities (54.7%-80.7%) with HTs proteins from various plants. DoHT1 belonged to the MST (monosaccharide transporter) group of the evolutionary tree, and was closely related to the Phalaenopsis equestris. DoHT1 was differentially expressed in the three included organs. The transcripts were significantly the most abundant in the leaves with 19.36 fold than roots, then 1.82 fold in the stems than the roots. The identification and molecular characterization of the full length DoHT1 will be essential for further function study of the gene during the regulation of sugar metabolism of D. officinale.

Critical Care Resources in Guangdong Province of China: Three Surveys from 2005 to 2015.

OBJECTIVES: Data about the critical care resources in China remain scarce. The purpose of this study was to investigate the variation and distribution of critical care resources in Guangdong province from 2005 to 2015. DESIGN: Data in regard to critical care resources were collected through questionnaires and visits every 5 years from 2005. SETTING: All hospitals in Guangdong province were screened and hospitals that provide critical care services were enrolled. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: One hundred eleven, 158, and 284 hospitals that provide critical care services were enrolled in the three consecutive surveys respectively. The number of ICUs, ICU beds, intensivists, and nurses increased to 324, 3,956, 2,470, and 7,695, respectively, by 2015. Adjusted by population, the number of ICU beds per 100,000 (100,000) population increased by 147.7% from 2005 to 2015, and the number of intensivists and nurses per 100,000 population increased by 35.3% and 55.1% from 2011 to 2015. However, the numbers in the Pearl River Delta, a richer area, were higher than those in the non-Pearl River Delta area (ICU beds: 4.64 vs 2.58; intensivists: 2.90 vs 1.61; nurses: 9.30 vs 4.71 in 2015). In terms of staff training, only 17.85% of intensivists and 14.29% of nurses have completed a formal accredited critical care training program by 2015. CONCLUSIONS: Our study was the first one to investigate the trend and distribution of critical care resources in China. The quantity of ICU beds and staff has been increasing rapidly, but professional training for staff was inadequate. The distribution of critical care resources was unbalanced. Our study can be beneficial for healthcare policymaking and the allocation of critical care resources in Guangdong province and other provinces in China.

A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR.

Clinical manifestations of severe enterovirus 71 infection and early assessment in a Southern China population.

BACKGROUND: Enterovirus 71 (EV-A71) shows a potential of rapid death, but the natural history of the infection is poorly known. This study aimed to examine the natural history of EV-A71 infection. METHODS: This was a prospective longitudinal observational study performed between January 1st and October 31st, 2012, at three hospitals in Guangdong, China. Subjects with positive EV-A71 RNA laboratory test results were included. Disease progression was documented with MRI, autopsies, and follow-up. Symptoms/signs with potential association with risk of death were analyzed. RESULTS: Among the 288 patients, neurologic symptoms and signs were observed (emotional movement disorders, dyskinesia, involuntary movements, autonomic dysfunction, and disturbance of consciousness). Some of them occurred as initial symptoms. Myoclonic jerks/tremors were observed among >50% of the patients; nearly 40% of patients presented fatigue and 25% were with vomiting. Twenty-eight patients (9.7%) presented poor peripheral perfusion within 53.4 ± 26.1 h; 23 patients (8.0%) presented pulmonary edema and/or hemorrhage within 62.9 ± 28.6 h. Seventeen (5.9%) patients were in a coma. Seven (2.4%) patients died within 62.9 ± 28.6 h. Seventy-seven survivors underwent head and spinal cord MRI and 37.7% (29/77) showed abnormalities. Two fatal cases showed neuronal necrosis, softening, perivascular cuffing, colloid, and neuronophagia phenomenon in the brainstem. CONCLUSIONS: Patients with EV-A71 infection showed high complexity of symptoms and onset timing. Death risk may be indicated by autokinetic eyeball, eyeball ataxia, severe coma, respiratory rhythm abnormality, absent pharyngeal reflex, ultrahyperpyrexia, excessive tachycardia, pulmonary edema and/or hemorrhage, and refractory shock and ataxic respiration. Early assessment of these symptoms/signs is important for proper management.

[Analysis of Applying Chinese Medical Clinical Pathway for Treating Attention-deficit Hyperactivity Disorder].

OBJECTIVE: To evaluate the application effect of Chinese medical clinical pathway for treating attention-deficit hyperactivity disorder (ADHD), and to provide evidence for further improving clinical pathways. METHODS: Totally 270 ADHD children patients were recruited and treated at pediatrics clinics of 9 cooperative hospitals from December 2011 to December 2012. The treatment course for all was 3 months. Scores of attention deficit and hyperactivity rating scale, scores of behavior, Conners index of hyperactivity (CIH), and Chinese medical syndrome scores were compared between before and after treatment. The efficacy difference in various sexes, ages, and disease courses were evaluated by judging standards for Chinese medical syndrome and ADHD. RESULTS: Fifteen children patients who entered clinical pathway dropped out, and the rest 255 completed this trial. Compared with before treatment, total scores of attention deficit and hyperactivity rating scale, scores of attention deficit and hyperactivity rating scale, CIH, and Chinese medical syndrome scores obviously decreased (all P < 0.01). The total effective rate in disease efficacy was 87.8% (224/255 cases), and the total effective rate in Chinese medical syndrome curative effect was 87.5% (223/255 cases). The clinical curative effect was not influenced by age, gender, or course of disease when statistically analyzed from judging standards for Chinese medical syndrome or for disease efficacy. CONCLUSION: Intervention by Chinese medical clinical pathway could improve ADHD patients' symptoms, and its efficacy was not influenced by sex, age, or course of disease.

[Cloning and expression analysis of a zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein encoding gene in Dendrobium officinale].

The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinale.

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.

[The principle of temperature measurement of a non-diffuse emitter].

In order to measure the temperature of the object in the case of non-diffuse emission within a finite solid angle, a new concept for radiation thermometry, the apparent emissivity, has been presented firstly after a proper mathematical transformation of the radiation measurement equation and its characteristics have also been investigated. The results indicated that although the apparent emissivity is complex in form, it is only the function of the wavelength for one measurement even if the object is non-diffuse. So the temperature of the object in the case of non-diffuse emission within a finite solid angle can be solved by modeling the apparent emissivity. Then the equations for monochromatic and wavelength radiation measurement were deduced. At the same time, the measurement of temperature for a finite area was investigated, and it can be found that if the temperatures of the finite area are the same, they can also be determined by modeling the apparent emissivity.

Comparative transcriptome analysis and identification of candidate R2R3-MYB genes involved in anthraquinone biosynthesis in Rheum palmatum L.

BACKGROUND: Rheum palmatum L. has important medicinal value because it contains biologically active anthraquinones. However, the key genes and TFs involved in anthraquinone biosynthesis and regulation in R. palmatum remain unclear. METHODS: Based on full length transcriptome data, in this study, we screened the differentially expressed genes in the anthraquinone biosynthesis pathway. The R2R3-MYB family genes of R. palmatum were systematically identified based on full-length transcriptome sequencing followed by bioinformatics analyses. The correlation analysis was carried out by using co-expression analysis, protein interaction analysis, and real-time fluorescence quantitative analysis after MeJA treatment. The RpMYB81 and RpMYB98 genes were amplified by RT-PCR, and their subcellular localization and self-activation characteristics were analyzed. RESULTS: Comparative transcriptome analysis results revealed a total of 3525 upregulated and 6043 downregulated DEGs in the CK versus MeJA group; 28 DEGs were involved in the anthraquinone pathway. Eleven CHS genes that belonged to the PKS family were differentially expressed and involved in anthraquinone biosynthesis. Twelve differentially expressed MYBs genes were found to be co-expressed and interact with CHS genes. Furthermore, 52 MYB genes were identified as positive regulators of anthraquinone biosynthesis and were further characterized. Three MYB genes including RpMYB81, RpMYB98, and RpMYB100 responded to MeJA treatment in R. palmatum, and the levels of these genes were verified by qRT-PCR. RpMYB81 was related to anthraquinone biosynthesis. RpMYB98 had an interaction with genes in the anthraquinone biosynthesis pathway. RpMYB81 and RpMYB98 were mainly localized in the nucleus. RpMYB81 had self-activation activity, while RpMYB98 had no self-activation activity. CONCLUSION: RpMYB81, RpMYB98, and RpMYB100 were significantly induced by MeJA treatment. RpMYB81 and RpMYB98 are located in the nucleus, and RpMYB81 has transcriptional activity, suggesting that it might be involved in the transcriptional regulation of anthraquinone biosynthesis in R. palmatum.

[Observations of in vitro pollen germination of Prunella vulgaris].

To determine the optimal condition of pollen germination. The pollen of Prunella vulgaris was cultured in vitro. Pollen germination rates were recorded using 10% H3BO4, 30% Ca(NO3)2, 20% MgSO4 and 10% KNO3 as the basic mineral medium with PEG of different molecular weight, sucrose of various density and multiple pH value. The rates were also measured under different cultivation temperature and pollen acquisition time. The optimal condition of pollen germination is 10% H3 BO4, 30% Ca(NO3)2, 20% MgSO4, 10% KNO3, and 25% PEG-4000 as the medium, with pH about 6. 5 and pollen acquired at the beginning of blossom.