Conservation Strategies for Aquilaria sinensis: Insights from DNA Barcoding and ISSR Markers.

The evergreen tree species Aquilaria sinensis holds significant economic importance due to its specific medicinal values and increasing market demand. However, the unrestricted illegal exploitation of its wild population poses a threat to its survival. This study aims to contribute to the conservation efforts of A. sinensis by constructing a library database of DNA barcodes, including two chloroplast genes (psbA-trnH and matK) and two nuclear genes (ITS and ITS2). Additionally, the genetic diversity and structure were estimated using inter-simple sequence repeats (ISSR) markers. Four barcodes of 57 collections gained 194 sequences, and 1371 polymorphic bands (98.63%) were observed using DNA ISSR fingerprinting. The Nei's gene diversity (H) of A. sinensis at the species level is 0.2132, while the Shannon information index (I) is 0.3128. The analysis of molecular variance revealed a large significant proportion of total genetic variations and differentiation among populations (Gst = 0.4219), despite a relatively gene flow (Nm = 0.6853) among populations, which were divided into two groups by cluster analysis. There was a close genetic relationship among populations with distances of 0.0845 to 0.5555. This study provides evidence of the efficacy and dependability of establishing a DNA barcode database and using ISSR markers to assess the extent of genetic diversity A. sinensis. Preserving the genetic resources through the conservation of existing populations offers a valuable proposition. The effective utilization of these resources will be further deliberated in subsequent breeding endeavors, with the potential to breed agarwood commercial lines.

Analysis of the chloroplast genome and phylogenetic evolution of Bidens pilosa.

Chloroplast genomes for 3 Bidens plants endemic to China (Bidens bipinnata Linn., Bidens pilosa Linn., and Bidens alba var. radiata) have been sequenced, assembled and annotated in this study to distinguish their molecular characterization and phylogenetic relationships. The chloroplast genomes are in typical quadripartite structure with two inverted repeat regions separating a large single copy region and a small single copy region, and ranged from 151,599 to 154,478 bp in length. Similar number of SSRs and long repeats were found in Bidens, wherein mononucleotide repeats (A/T), forward and palindromic repeats were the most in abundance. Gene loss of clpP and psbD, IR expansion and contraction were detected in these Bidens plants. It seems that ndhE, ndhF, ndhG, and rpl32 from the Bidens plants were under positive selection while the majority of chloroplast genes were under purifying selection. Phylogenetic analysis revealed that 3 Bidens plants clustered together and further formed molophyletic clade with other Bidens species, indicating Bidens plants might be under radiation adaptive selection to the changing environment world-widely. Moreover, mutation hotspot analysis and in silico PCR analysis indicated that inter-genic regions of ndhD-ccsA, ndhI-ndhG, ndhF-rpl32, trnL_UAG-rpl32, ndhE-psaC, matK-rps16, rps2-atpI, cemA-petA, petN-psbM were candidate markers of molecular identification for Bidens plants. This study may provide useful information for genetic diversity analysis and molecular identification for Bidens species.

A Cohort Study on Deficiency of ADA2 from China.

PURPOSE: Deficiency of adenosine deaminase 2 (DADA2), an autosomal recessive autoinflammatory disorder caused by biallelic loss-of-function variants in adenosine deaminase 2 (ADA2), has not been systemically investigated in Chinese population yet. We aim to further characterize DADA2 cases in China. METHODS: A retrospective analysis of patients with DADA2 identified through whole exome sequencing (WES) at seventeen rheumatology centers across China was conducted. Clinical characteristics, laboratory findings, genotype, and treatment response were analyzed. RESULTS: Thirty patients with DADA2 were enrolled between January 2015 and December 2021. Adenosine deaminase 2 enzymatic activity was low in all tested cases to confirm pathogenicity. Median age of disease presentation was 4.3 years and the median age at diagnosis was 7.8 years. All but one patient presented during childhood and two subjects died from complications of their disease. The patients most commonly presented with systemic inflammation (92.9%), vasculitis (86.7%), and hypogammaglobinemia (73.3%) while one patient presented with bone marrow failure (BMF) with variable cytopenia. Twenty-three (76.7%) patients were treated with TNF inhibitors (TNFi), while two (6.7%) underwent hematopoietic stem cell transplantation (HSCT). They all achieved clinical remission. A total of thirty-nine ADA2 causative variants were identified, six of which were novel. CONCLUSION: To establish early diagnosis and improve clinical outcomes, genetic screening and/or testing of ADA2 enzymatic activity should be performed in patients with suspected clinical features. TNFi is considered as first line treatment for those with vascular phenotypes. HSCT may be beneficial for those with hematological disease or in those who are refractory to TNFi.

Comparative chloroplast genome analyses of Amomum: insights into evolutionary history and species identification.

BACKGROUND: Species in genus Amomum always have important medicinal and economic values. Classification of Amomum using morphological characters has long been a challenge because they exhibit high similarity. The main goals of this study were to mine genetic markers from cp genomes for Amomum species identification and discover their evolutionary history through comparative analysis. RESULTS: Three species Amomum villosum, Amomum maximum and Amomum longipetiolatum were sequenced and annotated for the complete chloroplast (cp) genomes, and the cp genomes of A. longipetiolatum and A. maximum were the first reported. Three cp genomes exhibited typical quadripartite structures with 163,269-163,591 bp in length. Each genome encodes 130 functional genes including 79 protein-coding, 26 tRNAs and 3 rRNAs genes. 113-152 SSRs and 99 long repeats were identified in the three cp genomes. By designing specific primers, we amplified the highly variable loci and the mined genetic marker ccsA exhibited a relatively high species identification resolution in Amomum. The nonsynonymous and synonymous substitution ratios (Ka/Ks) in Amomum and Alpinia showed that most genes were subjected to a purifying selection. Phylogenetic analysis revealed the evolutionary relationships of Amomum and Alpinia species and proved that Amomum is paraphyletic. In addition, the sequenced sample of A. villosum was found to be a hybrid, becoming the first report of natural hybridization of this genus. Meanwhile, the high-throughput sequencing-based ITS2 analysis was proved to be an efficient tool for interspecific hybrid identification and with the help of the chloroplast genome, the hybrid parents can be also be determined. CONCLUSION: The comparative analysis and mined genetic markers of cp genomes were conducive to species identification and evolutionary relationships of Amomum.

DNA barcode reference library construction and genetic diversity and structure analysis of Amomum villosum Lour. (Zingiberaceae) populations in Guangdong Province.

BACKGROUND: Amomum villosum Lour. is the plant that produces the famous traditional Chinese medicine Amomi Fructus. Frequent habitat destruction seriously threatens A. villosum germplasm resources. Genetic diversity is very important to the optimization of germplasm resources and population protection, but the range of inherited traits within A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library as a resource for conservation efforts. METHODS: DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and used in the construction of a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei's gene diversity index (H), and Shannon's polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by measuring Nei's gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs), and gene flow (Nm). Analysis of molecular variance (AMOVA), Mantel tests, unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, and principal co-ordinates (PCoA) analysis were used to elucidate the genetic differentiation and relationship among populations. RESULTS: A total of 531 sequences were obtained from the five DNA barcodes with no variable sites from any of the barcode sequences. A total of 66 ISSR bands were generated from A. villosum populations using the selected six ISSR primers; 56 bands, 84.85% for all the seven A. villosum populations were polymorphic. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895), whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there is obvious genetic differentiation amongA. villosum populations and more genetic variations existed within each population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347.

Phenotypic variability in a child with Felty's syndrome: a case report.

BACKGROUND: Felty's syndrome (FS) is characterized by the triad of rheumatoid arthritis (RA), splenomegaly and neutropenia. The arthritis is typically severe and virtually always associated with high-titer rheumatoid factor. The presence of persistent neutropenia is generally required to make the diagnosis. Most patients diagnosed with FS are aged 50-70 years and have had RA for more than 10 years. It is rarely seen in patients with juvenile idiopathic arthritis (JIA), with only five cases having been reported throughout the world. CASE PRESENTATION: The present study describes the case of a 14-year-old female with a seven-year history of polyarticular JIA, presenting with splenomegaly, hepatomegaly, cholestasis and thrombocytopenia. However, she occasionally developed neutropenia. Titers of rheumatoid factor and anti-CCP were persistently high, and the antinuclear antibody titer was 1:320, while the antibody results for anti-dsDNA and anti-Sm were negative. Serum levels of IgA, IgG, IgM and IgE were all persistently elevated, and the ratio of CD19+ lymphocytes in the subgroups of lymphocytes was persistently high. The level of complements was normal. No STAT3 and STAT5B mutations were found by next-generation sequencing. The patient did not respond to methotrexate, prednisolone, hydroxychloroquine (HCQ), sulfasalazine and etanercept but was responsive to rituximab. CONCLUSIONS: JIA, thrombocytopenia and splenomegaly are the most common and important features in six children with FS, while persistent neutropenia is not seen in all these patients. No complement deficiency has been found in children with FS so far. Manifestations of FS without neutropenia may be extremely rare. There are differences between adults and children in the clinical and laboratory features of FS.

Dural sinus thrombosis and giant pulmonary artery aneurysm in paediatric Behçet's disease.

Paediatric Behçet's disease (BD) accounts for only 2-5% of all patients with BD. Neurological and vascular involvement account for only 3.6% and 1.8% of paediatric BD in China, but both are lethal complications. We report the case of a 12-year-old Chinese boy presenting with recurrent oral ulcers, extensive thrombosis, cerebral sinus vein thrombosis and bilateral inferior pulmonary artery aneurysm. With treatment that included oral prednisone, a monthly pulse of cyclophosphamide followed by mycophenolate mofetil, and anticoagulant therapy, the patient became symptom free, his C-reactive protein and erythrocyte sedimentation rate remained normal, and the right inferior pulmonary artery aneurysm was reduced to normal. However, the left inferior pulmonary artery aneurysm progressively expanded to 64.9 mm×36.2 mm×44 mm. Eventually, the patient underwent left pulmonary aneurysm resection and a left inferior lobectomy. The post-operative maintenance treatment included oral prednisone, mycophenolate mofetil and low-dose aspirin, and the patient was followed for 2 years without recurrence. Additionally, we retrospectively analysed the clinical characteristics of 23 paediatric BD patients from our medical centre and briefly reviewed the literature on paediatric BD.

Determination of 5-nitro-2-furaldehyde as marker residue for nitrofurazone treatment in farmed shrimps and with addressing the use of a novel internal standard.

We developed a significantly improved ultra-high performance liquid chromatography-tandem mass spectrometry method for determination of 5-nitro-2-furaldehyde (NF) as a surrogate using a novel internal standard for the detection of nitrofurazone. We used 2,4-dinitrophenylhydrazine derivatization and furfural as the internal standard. Derivatization was easily performed in HCl using ultrasonic manipulation for 5 min followed by liquid extraction using ethyl acetate. The samples were concentrated and purified using reverse phase and alumina cartridges in tandem. The derivatives were separated using a linear gradient elution on a C18 column with methanol and water as the mobile phase in negative ionization mode and multiple reaction monitoring. Under the optimized conditions, the calibration curves were linear from 0.2 to 20 µg/L with correlation coefficients >0.999. Mean recoveries were 80.8 to 104.4% with the intra- and inter-day relative standard deviations <15% at spiking levels of 0.1 to 10 µg/kg. The limits of detection and quantification were 0.05 and 0.1 µg/kg, respectively. This method is a robust tool for the identification and quantitative determination of NF in shrimp samples.

Expanding the spectrum of A20 haploinsufficiency in two Chinese families: cases report.

BACKGROUND: The association between mutations in the TNFAIP3 gene and a new autoinflammatory disease (called A20 haploinsufficiency, HA20) has recently been recognized. Here, we describe four patients with HA20 from two unrelated Chinese families. CASE PRESENTATION: A total of four patients from two families were included. The average age at onset was 5.9 years. All patients had no signs of eye or skin problems, such as uveitis, rash, folliculitis and dermal abscess. Prior to the recognition of HA20, P1 was diagnosed with SLE, liver fibrosis and hypothyroidism. She also had no oral, genital or perineal ulcers. P2 was diagnosed with Crohn's disease and inflammatory bowel disease-related arthritis (IBD-RA). He had a perianal abscess but no oral or genital ulcers. P3, the father of P1 and P2, only had mild oral ulcers, arthralgia, and archosyrinx. P4 was diagnosed with polyarticular juvenile idiopathic arthritis (JIA), macrophage activation syndrome (MAS) and interstitial lung disease (ILD). Whole exome sequencing (WES) was performed in two families. WES revealed heterozygous c.559C > T in the TNFAIP3 gene in P1, P2 and P3, while the c.259C > T mutation in the TNFAIP3 gene was identified in P4. The c.259C > T mutations is novel. CONCLUSION: HA20 had a different phenotype between families and even between family members with the same mutation. Liver fibrosis, hypothyroidism, ILD and MAS in the patients with HA20 were first reported in this study. Our results expanded the phenotype and genotype spectrum of A20 haploinsufficiency.

A mutation in PIK3CD gene causing pediatric systemic lupus erythematosus: A case report.

RATIONALE: Gain of function (GOF) mutations in PIK3CD gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, cytomegalovirus and/or epstein-Barr virus (EBV) viremia, and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. PATIENT CONCERNS: We report a patient who was diagnosed with systemic lupus erythematosus (SLE) at a young age and was recently found to carry heterozygous mutations in PIK3CD. The patient not only presented with recurrent sinopulmonary infections, CD4 lymphopenia, lymphadenopathy, EBV viremia, and elevated serum IgM, but also met classification criteria of SLE based on persistent proteinuria and hematuria, leukopenia and anemia, low level of serum complement, and positive autoantibody for antinuclear antibodies. DIAGNOSES: Activated PI3Kδ syndrome. INTERVENTIONS: Oral prednisolone and hydroxychloroquine combined with mycophenolate mofetil was given to the patient. He was currently receiving intravenous immunoglobulin per month in association with hydroxychloroquine, low-dose prednisolone, and mycophenolate mofetil. OUTCOMES: At present, the level of complement restored to normal, hematuria and proteinuria disappeared, and liver function returned to normal. LESSONS: SLE may be a novel phenotype of GOF mutation in PI3CKD gene (GOF PIK3CD).

Heat shock protein 70-2 and tumor necrosis factor-α gene polymorphisms in Chinese children with Henoch-Schönlein purpura.

BACKGROUND: Henoch-Schönlein purpura (HSP) or IgA-associated vasculitis is related to immune disturbances. Polymorphisms of the heat shock protein 70-2 gene (HSP70-2) and the tumor necrosis factor-a gene (TNF-α) are known to be associated with immune diseases. The purpose of this study was to investigate the likely association of HSP70-2 (+1267A/G) and TNF-α (+308A/G) gene polymorphisms with HSP in children. METHODS: The polymerase chain reaction restriction fragment length polymorphism method was used to detect the HSP70-2 and TNF-α polymorphisms in 205 cases of children with HSP and 53 controls; and the association of these polymorphisms with HSP and HSP nephritis (HSPN) was analyzed. RESULTS: The G/G genotypic frequencies at the +1267A/G position of HSP70-2 in the HSP group (22.9%) were significantly higher than those in the healthy control group (9.4%) (χ(2)=4.764, P<0.05). The frequencies of the A/A, A/G and G/G genotypes of HSP70-2 in patients in the nephritis-free group and the HSPN group showed no statistically significant difference. The A/A genotype frequency at the +308G/A position of TNF-α in the HSP group was 8.3%, which was higher than that in the control group (χ(2)=6.447, P<0.05). The A allele frequency of TNF-α in the HSP group was higher than that in the control group, with a statistically significant difference (χ(2)=7.241, P<0.05). CONCLUSIONS: The HSP70-2 (+1267A/G) and TNF-α (+308G/A) gene polymorphisms were associated with HSP in children. The G/G homozygosity of HSP70-2 and the A/A homozygosity of TNF-α may be genetic predisposing factors for HSP.

Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4.

Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45°C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.

Characterization and synthetic application of a novel beta1,3-galactosyltransferase from Escherichia coli O55:H7.

A beta1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion, either Mn(2+) or Mg(2+), for its activity and is active between pH 6.0-8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase, benzyl beta-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility of WbgO.