Characterization of the Heat Shock Transcription Factor Family in Lycoris radiata and Its Potential Roles in Response to Abiotic Stresses.

Heat shock transcription factors (HSFs) are an essential plant-specific transcription factor family that regulates the developmental and growth stages of plants, their signal transduction, and their response to different abiotic and biotic stresses. The HSF gene family has been characterized and systematically observed in various species; however, research on its association with Lycoris radiata is limited. This study identified 22 HSF genes (LrHSFs) in the transcriptome-sequencing data of L. radiata and categorized them into three classes including HSFA, HSFB, and HSFC, comprising 10, 8, and 4 genes, respectively. This research comprises basic bioinformatics analyses, such as protein sequence length, molecular weight, and the identification of its conserved motifs. According to the subcellular localization assessment, most LrHSFs were present in the nucleus. Furthermore, the LrHSF gene expression in various tissues, flower developmental stages, two hormones stress, and under four different abiotic stresses were characterized. The data indicated that LrHSF genes, especially LrHSF5, were essentially involved in L. radiata development and its response to different abiotic and hormone stresses. The gene-gene interaction network analysis revealed the presence of synergistic effects between various LrHSF genes' responses against abiotic stresses. In conclusion, these results provided crucial data for further functional analyses of LrHSF genes, which could help successful molecular breeding in L. radiata.

Functional Characterization of AP2/ERF Transcription Factors during Flower Development and Anthocyanin Biosynthesis Related Candidate Genes in Lycoris.

The APETALA2/ethylene-responsive transcription factor (AP2/ERF) family has been extensively investigated because of its significant involvement in plant development, growth, fruit ripening, metabolism, and plant stress responses. To date, there has been little investigation into how the AP2/ERF genes influence flower formation and anthocyanin biosynthesis in Lycoris. Herein, 80 putative LrAP2/ERF transcription factors (TFs) with complete open reading frames (ORFs) were retrieved from the Lycoris transcriptome sequence data, which could be divided into five subfamilies dependent on their complete protein sequences. Furthermore, our findings demonstrated that genes belonging to the same subfamily had structural similarities and conserved motifs. LrAP2/ERF genes were analyzed for playing an important role in plant growth, water deprivation, and flower formation by means of gene ontology (GO) enrichment analysis. The expression pattern of the LrAP2/ERF genes differed across tissues and might be important for Lycoris growth and flower development. In response to methyl jasmonate (MeJA) exposure and drought stress, the expression of each LrAP2/ERF gene varied across tissues and time. Moreover, a total of 20 anthocyanin components were characterized using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis, and pelargonidin-3-O-glucoside-5-O-arabinoside was identified as the major anthocyanin aglycone responsible for the coloration of the red petals in Lycoris. In addition, we mapped the relationships between genes and metabolites and found that LrAP2/ERF16 is strongly linked to pelargonidin accumulation in Lycoris petals. These findings provide the basic conceptual groundwork for future research into the molecular underpinnings and regulation mechanisms of AP2/ERF TFs in anthocyanin accumulation and Lycoris floral development.

Clinical application of SARS-CoV-2 antibody detection and monoclonal antibody therapies against COVID-19.

The purpose of this study was to investigate the clinical application of severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) specific antibody detection and anti-SARS-CoV-2 specific monoclonal antibodies (mAbs) in the treatment of coronavirus infectious disease 2019 (COVID-19). The dynamic changes of SARS-CoV-2 specific antibodies during COVID-19 were studied. Immunoglobulin M (IgM) appeared earlier and lasted for a short time, while immunoglobulin G (IgG) appeared later and lasted longer. IgM tests can be used for early diagnosis of COVID-19, and IgG tests can be used for late diagnosis of COVID-19 and identification of asymptomatic infected persons. The combination of antibody testing and nucleic acid testing, which complement each other, can improve the diagnosis rate of COVID-19. Monoclonal anti-SARS-CoV-2 specific antibodies can be used to treat hospitalized severe and critically ill patients and non-hospitalized mild to moderate COVID-19 patients. COVID-19 convalescent plasma, highly concentrated immunoglobulin, and anti-SARS-CoV-2 specific mAbs are examples of anti-SARS-CoV-2 antibody products. Due to the continuous emergence of mutated strains of the novel coronavirus, especially omicron, its immune escape ability and infectivity are enhanced, making the effects of authorized products reduced or invalid. Therefore, the optimal application of anti-SARS-CoV-2 antibody products (especially anti-SARS-CoV-2 specific mAbs) is more effective in the treatment of COVID-19 and more conducive to patient recovery.

Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata.

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I-III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

Association of mitochondrial respiratory chain enzymes with the risk and mortality of sepsis among Chinese children.

BACKGROUND: Sepsis is a leading cause of pediatric morbidity and mortality worldwide. The aim of this study was to explore the association of decreased mitochondrial respiratory chain enzyme activities with the risk for pediatric sepsis, and explore their association with mortality among affected children. METHODS: A total of 50 incident cases with sepsis and 49 healthy controls participated in this study. The level of serum coenzyme Q10 was measured by high-performance liquid chromatography, and selected mitochondrial respiratory chain enzymes in WBC were measured using spectrophotometric. Logistic regression models were used to estimate odds ratio (OR) and 95% confidence interval (CI). RESULTS: The levels of CoQ10, complex II, complex I + III and FoF1-ATPase were significantly higher in healthy controls than in children with sepsis (p < 0.001, = 0.004, < 0.001 and < 0.001, respectively). In children with sepsis, levels of CoQ10 and complex I + III were significantly higher in survived cases than in deceased cases (p < 0.001). Per 0.05 µmol/L, 50 nmol/min.mg and 100 nmol/min.mg increment in CoQ10, complex I + III and FoF1-ATPase were associated with significantly lowered risk of having sepsis, even after adjusting for confounding factors (OR = 0.85, 0.68 and 0.04, p = 0.001, < 0.001 and < 0.001, respectively). Per 0.05 µmol/L and 50 nmol/min.mg increment in CoQ10 and complex I + III was associated with significantly lowered risk of dying from sepsis during hospitalization, and significance retained after adjustment (OR = 0.73 and 0.76, 95% CI: 0.59 to 0.90 and 0.64 to 0.89, p = 0.004 and 0.001, respectively) in children with sepsis. CONCLUSIONS: Our findings indicate the promising predictive contribution of low serum CoQ10 and complex I + III to the risk of pediatric sepsis and its associated mortality during hospitalization among Chinese children. Trial registration The trial was registered with www.chictr.org.cn , number ChiCTR-IOR-15006446 on May 05, 2015. Retrospectively registered.

Enhancement of a recombinase-aided amplification assay using betaine and pullulan.

Background: Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) was initially explored. Methods: Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time and fluorescence value, and the basic method was characterized by 2% agarose gel electrophoresis. Results: Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2 M, 0.4 M, and 0.6 M betaine and 10% pullulan could enhance the RAA. The new RAA assays with betaine and pullulan were named B-RAA and P-RAA, respectively. Using the B-RAA and P-RAA fluorescence methods, the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes, respectively, and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv, respectively. Using the basic method, the sensitivity could be increased 10-fold. We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 102 copies/µL (compared with 103 copies/µL in the RAA assay). Conclusions: Thus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.

Internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of human papillomavirus genotypes 16 and 18 using extracted DNA and samples treated with nucleic acid releasing agent.

Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 ℃ and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/µL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/µL for HPV16 and 100 copies/µL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.

Development of a duplex reverse transcription recombinase-aided amplification assay for respiratory syncytial virus incorporating an internal control.

Human respiratory syncytial virus (RSV) is a common viral pathogen that causes lower respiratory tract infections in infants and children globally. In this study, we developed a duplex reverse transcription recombinase-aided amplification (duplex-rtRAA) assay containing an internal control in a single closed tube for the detection of human RSV. The internal control in the amplification effectively eliminated false-negative results and ensured the accuracy of the duplex-rtRAA system. We first developed and evaluated a universal singleplex-rtRAA assay for RSV. The sensitivity of this assay for RSV was determined as 4.4 copies per reaction, and the specificity was 100%. Next, a duplex-rtRAA assay with an internal control was established. The sensitivity of the duplex-rtRAA assay approached 5.0 copies per reaction, and no cross-reaction with other common respiratory viruses was observed. The two detection methods (singleplex-rtRAA and duplex-rtRAA) developed in this study were used to test 278 clinical specimens, and the results showed absolute consistency with RSV RT-qPCR analysis, demonstrating 100% diagnostic sensitivity and specificity. These data indicate that the duplex-rtRAA has great potential for the rapid detection of RSV with a high sensitivity.

Layer-by-Layer Heparinization of the Cell Surface by Using Heparin-Binding Peptide Functionalized Human Serum Albumin.

Layer-by-layer heparinization of therapeutic cells prior to transplantation is an effective way to inhibit the instant blood-mediated inflammatory reactions (IBMIRs), which are the major cause of early cell graft loss during post-transplantation. Here, a conjugate of heparin-binding peptide (HBP) and human serum albumin (HSA), HBP-HSA, was synthesized by using heterobifunctional crosslinker. After the first heparin layer was coated on human umbilical vein endothelial cells (HUVECs) by means of the HBP-polyethylene glycol-phospholipid conjugate, HBP-HSA and heparin were then applied to the cell surface sequentially to form multiple layers. The immobilization and retention of heparin were analyzed by confocal microscopy and flow cytometry, respectively, and the cytotoxity of HBP-HSA was further evaluated by cell viability assay. Results indicated that heparin was successfully introduced to the cell surface in a layer-by-layer way and retained for at least 24 h, while the cytotoxity of HBP-HSA was negligible at the working concentration. Accordingly, this conjugate provides a promising method for co-immobilization of heparin and HSA to the cell surface under physiological conditions with improved biocompatibility.

Association between serum 25-hydroxyvitamin D concentration and pulmonary infection in children.

We assessed the relationship between serum 25-hydroxyvitamin D (25(OH)D) level and community-acquired pneumonia (CAP) among Chinese children.This observational study examined children aged 3 days to 14 years (n = 1582) from the Capital Institute of Pediatrics in 2009 to 2011. There were 797 children in the CAP group and 785 controls. The CAP group was divided into 2 groups: a pneumonia group and pneumonia-induced sepsis group. The serum 25(OH)D level was estimated using micro whole blood chemiluminescence.The average serum 25(OH)D level in all samples was 25.32 ±â€Š14.07 ng/mL, with the CAP group showing a lower value than the control group (P < .001). There were also significant differences between the pneumonia group and pneumonia-induced sepsis group (P < .001). In the pneumonia-induced sepsis group, significant differences in serum 25(OH)D levels were observed in children who received mechanical ventilation or presenting with multiple organ dysfunction (P < .01).All serum 25(OH)D levels in the pneumonia group and pneumonia-induced sepsis group were below normal levels, particularly in the sepsis group. A lower serum 25(OH)D level was associated with more serious symptoms in CAP children. Children with low serum 25(OH)D levels may be at higher risk of receiving mechanical ventilation and presenting with multiple organ dysfunction. These findings suggest that vitamin D supplements are beneficial for the treatment and prevention of CAP.

The association of endothelial nitric oxide synthase gene single nucleotide polymorphisms with paediatric systemic lupus erythematosus.

OBJECTIVES: Endothelial nitric oxide synthase (eNOS) is a type of nitric oxide synthase that mainly exists in the endothelium. It produces nitric oxide (NO) to regulate the function of endothelial cells. Endothelial dysfunction and increased NO metabolites have been shown in animal models of lupus and in lupus patients, so eNOS gene polymorphisms may be important in the pathogenesis of SLE. This study aimed to investigate the association of the single nucleotide polymorphisms (SNPs) of eNOS and paediatric systemic lupus erythematosus (pSLE). METHODS: A total of 91 pSLE cases and 90 healthy controls were used in this study. We divided these patients into 4 subgroups according to kidney or central nervous system involvement. Four selected SNPs in the gene were analysed with MALDI-TOF mass spectrometry. Statistical methods were carried out to investigate the correlation between the SNPs and pSLE. RESULTS: SNP rs1808593 genotype GT in case group were significantly higher than those in the control group (p=0.047), and the genotype GT had positive correlation with pSLE (OR=1.93, 95% CI: 1.01-3.69). In subgroup C (the patients with central nervous system but no kidney damage), the genotype GT was significantly higher than those in the control group (p=0.028), and the genotype GT was related to pSLE with central nervous system damage (OR=6.24, 95% CI: 1.17-33.15). In male patients, we found SNP rs1808593 genotype GT in pSLE group was significantly higher than in the control group (p=0.0065), and the risk of pSLE increased in patients with genotype GT (OR=8.36, 95% CI: 2.02-34.6). CONCLUSIONS: SNP rs1808593 GT genotype is significantly higher than that in the control group, which may indicate that this genotype increases the risk of pSLE, especially in boys, and also this genotype might increase the risk of central nervous system involvement. Therefore, eNOS gene SNP rs1808593 genotype may have an important role in predicting the occurrence of pSLE and central nervous system complications in pSLE.

Design and synthesis of novel 3-sulfonylpyrazol-4-amino pyrimidines as potent anaplastic lymphoma kinase (ALK) inhibitors.

Anaplastic lymphoma kinase (ALK) is a highly attractive therapeutic target for the treatment of some non-small cell lung cancer patients. This Letter describes the further SAR exploration on the novel 3-sulfonylpyrazol-4-amino pyrimidine scaffold. This work identified a compound 53 with very good in vitro/in vivo efficacies, good DMPK properties together with better hERG tolerability and it is currently being profiled for the evaluation as a potential pre-clinical candidate.

[Vitamin D level in children with bloodstream infection].

OBJECTIVE: To investigate the difference in serum 25(OH)D level between children with bloodstream infection and healthy children. METHODS: A case-control study was conducted among 60 children with bloodstream infection who were hospitalized between January 2010 and December 2013 and had positive results of two blood cultures. Meanwhile, 60 aged-matched healthy children who underwent physical examination during the same period of time were enrolled as the healthy control group. Chemiluminescence was applied to measure the serum 25(OH)D level, and the constituent ratios of children with different serum 25(OH)D levels were compared between the two groups. RESULTS: The bloodstream infection group had a significantly lower serum 25(OH)D level than the healthy control group (P<0.01). Compared with the healthy control group, the bloodstream group had significantly lower constituent ratios of children with normal Vitamin D level (8% vs 35%) or vitamin D insufficiency (22% vs 43%) (P<0.05). Compared with the healthy control group, the bloodstream group had significantly higher constituent ratios of children with vitamin D deficiency (42% vs 13%) or severely vitamin D deficiency (28% vs 8%) (P<0.01). CONCLUSIONS: Vitamin D insufficiency prevails among children, and children with bloodstream infection have a significantly lower serum 25(OH)D level than healthy children.

Clinical features and risk factors for blood stream infections of Candida in neonates.

Candida species are the leading cause of invasive fungal infections in children admitted to hospital. However, few data exist with regard to the clinical features, risk factors and prognosis for candidemia in neonates. The present retrospective study included 40 neonates from the Affiliated Children's Hospital of the Capital Institute of Pediatrics (Beijing, China) in the time period between January 1, 2006 and December 31, 2010 (candidemia group, n=19; non-candidemia group, n=21). The clinical characteristics, prognosis and previously identified risk factors for the two groups were recorded. According to the forward stepwise multivariate logistic regression analysis, administration of antibiotics >2 weeks prior, the use of glycopeptide antibiotics, maternal candidal vaginitis and secondary gastrointestinal surgery were identified as predictors of candidiasis. When compared with the non-gastrointestinal dysfunction group, the proportion of neonates that had been subjected to parenteral nutrition, central venous catheters, gastrointestinal surgery, secondary gastrointestinal surgery, repeated tracheal intubation and glycopeptide antibiotic administration was significantly higher in the gastrointestinal dysfunction group (P<0.05). Long-term application of antibiotics, use of glycopeptide antibiotics, maternal candidal vaginitis and secondary gastrointestinal surgery appeared to be the risk factors of candidemia in neonates. The neonates co-existed with gastrointestinal dysfunction suffering from candidemia were likely to experience growth retardation at 6 months after hospital discharge. Candidemia is potentially life-threatening situation for neonates, and if patients do not succumb it may affect their early development.

Discovery of 2-arylamino-4-(1-methyl-3-isopropylsulfonyl-4-pyrazol-amino)pyrimidines as potent anaplastic lymphoma kinase (ALK) inhibitors.

A new series of 2,4-diamino pyrimidine derivatives with a sulfone-substituted pyrazole right side-chain were discovered as potent anaplastic lymphoma kinase inhibitors. Structure-activity relationship of the left side-chain on phenyl substitutions were explored which delivered many potent ALK inhibitors. Among them, 29a showed favorable pharmacokinetic profiles in rats and dogs together with significant antitumor efficacy in EML4-ALK fusion xenograft model.

Polymorphism rs2239185 in vitamin D receptor gene is associated with severe community-acquired pneumonia of children in Chinese Han population: a case-control study.

UNLABELLED: Vitamin D receptor (VDR) is a potential candidate gene for community-acquired pneumonia (CAP). Examining the susceptibility VDR gene for CAP is essential for early intervention, prevention of related complications, and improvement of outcome. A case-control study was performed to examine the association between rs2239185 of VDR gene and CAP among children in Chinese Han population. Polymerase chain reaction and direct sequencing were used to genotype rs2239185 in 91 CAP children and 94 healthy children. For rs2239185, individuals with TT genotype showed a significantly higher risk of CAP than those with CC plus CT genotypes (P = 0.008). The occurrence of T allele of rs2239185 was significantly more frequent in CAP children than those in normal controls (P = 0.045).We found through stratification analysis that CAP children with systemic inflammatory response syndrome (SIRS), leukocyte count (WBC) >10 × 10(9)/L, C-reactive protein (CRP) >25 mg/L, procalcitonin (PCT) >2 ng/mL, and pediatric critical illness score <80 scores showed significantly higher frequency of TT genotype than those in normal controls (P = 0.0012, 0.0035, 0.0005, 0.0002, and 0.0021, respectively). CONCLUSION: TT genotype of rs2239185 in VDR gene might be one of the potential genetic risk factors for CAP, and T allele of rs2239185 might be associated with the susceptibility to CAP and the severity of CAP.

High expression levels of microRNA-629, microRNA-525-5p and microRNA-516a-3p in paediatric systemic lupus erythematosus.

Paediatric systemic lupus erythematosus (pSLE) refers to childhood-onset systemic lupus erythematosus. pSLE has its own unique characteristics, and its pathogenesis is unclear. To study the relationship between microRNAs (miRNAs) and pSLE, we selected three pSLE patients who were newly diagnosed and had not yet been treated, and two controls were also included. We collected their peripheral blood mononuclear cells to perform Agilent human miRNA (8×15 k) 12.0 analysis. To verify the results, we next selected 12 other pSLE patients who had different disease activities and 3 healthy controls and conducted real-time PCR. The results showed high expression of miRNA-516a-3p, miRNA-629 and miRNA-525-5p in pSLE patients with active disease; these levels were normal in patients without active disease. Increased expression levels of these three miRNAs were positively correlated with the score obtained from the systemic lupus erythematosus disease activity index scoring system (SLEDAI) 2000 and C-reactive protein (CRP) levels. Furthermore, the target genes of these three miRNAs were important to the pathogenesis of pSLE. Therefore, these three miRNAs might be specific to pSLE and may be used as novel biomarkers of pSLE to diagnose and monitor the disease.

Asymmetric synthesis of α,ß-diamino acid derivates via Mannich-type reactions of a chiral Ni(II) complex of glycine with N-tosyl imines.

A practical procedure composed of an asymmetric Mannich-type reaction between N-tosyl imine and a Ni(II) complex of glycine with (R)-o-[N-(N-benzylprolyl)amino]bezaophenone (BPB) as a chiral auxiliary catalyzed by Et(3)N in DMF to (R,2R,3S)-complexes, and decomposition of products with HCl to offer syn-(2R,3S)-α,ß-diamino acids, was developed. Stereochemical mechanism of the Mannich reaction was proposed and supported by determining the absolute configuration of the product of the Mannich reaction relying on X-ray analysis. This two-step approach to amino acids was a general method and adapted to large-scale preparation.