Integrated metabolomics and transcriptomic analysis reveals metabolic changes of flavor compounds of Camellia assamica host plant after parasitized by Viscumarticulatum.

Tea is one of the most popular beverages, it has many health benefits and flavor properties due to the presence of numerous secondary metabolites. Camellia assamica is also a main source of tea, which is mainly planted in the regions of southwest China. In this study, a non-targeted and targeted metabolomics analysis and sensory evaluation on tea leaves with and without mistletoe (Viscum articulatum) was carried out using liquid chromatography-mass spectrometry. RNA-seq-based transcriptomic analysis was conducted in parallel on the same samples, subsequently gene expression and metabolic differentiation were also investigated. Tea leaves with mistletoe presented much lower contents of (-)-catechin, (-)-epicatechin, (-)-gallocatechin gallate and (-)-epicatechin gallate, but significantly higher levels of free amino acids including Arg, Asp, GABA and Gln than that without mistletoe. Transcriptomic analysis also confirmed the main differentially expressed genes (DEGs) containing phenylpropanoid and flavonoid biosynthesis were down-regulated, but genes of amino acid biosynthesis were up-regulated. qRT-PCR analysis further revealed that the relative expression of CsCHS, CsC4H, CsANS, CsLAR, and CsF3H was hindered, while CsglyA and CsilvE expression was increased.

Immunogenicity and immunoprotection of the functional TL-HN fragment derived from tetanus toxin.

Tetanus toxin (TeNT) is a protein toxin produced by Clostridium tetani bacteria, which causes hyperreflexia and rhabdomyolysis by spastic paralysis. Like botulinum neurotoxin, TeNT comprises a heavy chain (HC) and a light chain (LC) linked via an interchain disulfide bond, which include the following three functional domains: a receptor-binding domain (Hc), a translocation domain (HN), and a catalytic domain (LC). Herein, we produced and characterized three functional domains of TeNT and three types of TeNT-derived L-HN fragments (TL-HN, TL-GS-HN and TL-2A-HN), which contained L and HN domains but lacked the Hc domain. The immunological effects of these different functional domains or fragments of TeNT were explored in an animal model. Our investigations showed the TL-HN functional fragment provided the best immunoprotection among all the TeNT functional domains. The TL-HN fragment, as a protective antigen, induced the highest levels of neutralizing antibodies, indicating that it might contain some crucial epitopes. Further experiments revealed that the protective effect of TL-HN was superior to that of the THc, TL, or THN fragments, either individually or in combination. Therefore, the TL-HN fragment exerts an important function in immune protection against tetanus toxin, providing a good basis for the development of TeNT vaccines or antibodies, and could serve as a promising subunit vaccine to replace THc or tetanus toxoid (TT).

Tetanus toxin and botulinum neurotoxin-derived fusion molecules are effective bivalent vaccines.

Tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) are neuroprotein toxins, with the latter being the most toxic known protein. They are structurally similar and contain three functional domains: an N-terminal catalytic domain (light chain), an internal heavy-chain translocation domain (HN domain), and a C-terminal heavy chain receptor binding domain (Hc domain or RBD). In this study, fusion functional domain molecules consisting of the TeNT RBD (THc) and the BoNT/A RBD (AHc) (i.e., THc-Linker-AHc and AHc-Linker-THc) were designed, prepared, and identified. The interaction of each Hc domain and the ganglioside receptor (GT1b) or the receptor synaptic vesicle glycoprotein 2 (SV2) was explored in vitro. Their immune response characteristics and protective efficacy were investigated in animal models. The recombinant THc-linker-AHc and AHc-linker-THc proteins with the binding activity had the correct size and structure, thus representing novel subunit vaccines. THc-linker-AHc and AHc-linker-THc induced high levels of specific neutralizing antibodies, and showed strong immune protective efficacy against both toxins. The high antibody titers against the two novel fusion domain molecules and against individual THc and AHc suggested that the THc and AHc domains, as antigens in the fusion functional domain molecules, do not interact with each other and retain their full key epitopes responsible for inducing neutralizing antibodies. Thus, the recombinant THc-linker-AHc and AHc-linker-THc molecules are strong and effective bivalent biotoxin vaccines, protecting against two biotoxins simultaneously. Our experimental design will be valuable to develop recombinant double-RBD fusion molecules as potent bivalent subunit vaccines against bio-toxins. KEY POINTS: • Double-RBD fusion molecules from two toxins had the correct structure and activity. • THc-linker-AHc and AHc-linker-THc efficiently protected against both biotoxins. • Such bivalent biotoxin vaccines based on the RBD are a valuable experimental design.

Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase.

AIM: To evaluate the potential of two trabecular meshwork (TM)-specific promoters, Chitinase 3-like 1 (Ch3L1) and matrix gla protein (MGP), for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2 (scAAV2) vector technologies. METHODS: An scAAV2 vector with C3 transferase (C3) as the reporter gene (scAAV2-C3) was selected. The scAAV2-C3 vectors were driven by Ch3L1 (scAAV2-Ch3L1-C3), MGP (scAAV2-MGP-C3), enhanced MGP (scAAV2-eMGP-C3) and cytomegalovirus (scAAV2-CMV-C3), respectively. The cultured primary human TM cells were treated with each vector at different multiplicities of infections. Changes in cell morphology were observed by phase contrast microscopy. Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining, real-time quantitative polymerase chain reaction and Western blot. Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively. In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope. Intraocular pressure (IOP) was monitored using a rebound tonometer. Ocular responses were evaluated by slit-lamp microscopy. Ocular histopathology analysis was examined by hematoxylin and eosin staining. RESULTS: In TM cell culture studies, the vector-mediated C3 expression induced morphologic changes, disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rho-associated protein kinase signaling pathway. At the same dose, these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3, but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3. At low-injected dose, the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGP-C3-injected and scAAV2-eMGP-C3-injected eyes. At high-injected dose, significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes. Similar to scAAV2-CMV-C3, scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium. In anterior segment tissues of scAAV2-eMGP-C3-injected eyes, no obvious morphological changes were found except for the TM. Inflammation was absent. CONCLUSION: In scAAV2-transduced TM cells, the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus, but obviously higher than that of MGP. In the anterior chamber of rat eye, the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter, but not by Ch3L1 promoter. These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Novel homozygous ADAMTS17 missense variant in Weill-Marchesani syndrome.

AIM: To explore the phenotype and genotype of Weill-Marchesani syndrome (WMS) in a Chinese family and review related literature. METHODS: Three WMS patients and other unaffected individuals in this family with a history of consanguineous marriage were included in this study. Medical history, comprehensive ophthalmic examinations, and systemic evaluation, as well as whole exome and Sanger sequencing of specific genomic regions, were performed. RESULTS: The three affected siblings presented with short stature, brachydactyly and ocular disorders, including very shallow anterior chamber, high myopia, microspherophakia lens subluxation with stretched zonules and glaucoma. Genetic analysis verified a homozygous missense mutation (c.2983C>T: p. Arg995Trp) in ADAMTS17, which was correlated with the diseases in this family, indicating an autosomal recessive inherited manner of WMS. This review aims to summarize the mutation sites of WMS genes, so as to prevent the disease and better guide clinical diagnosis and treatment. CONCLUSION: A novel homozygous missense variant of ADAMTS17 is identified in a WMS family with a history of consanguineous marriage. Our study expands the range of mutations associated with WMS and deepens our understanding of pathology in disease associated with ADAMTS17 variants.

[Characteristics of Soil Carbon, Nitrogen, Phosphorus, and Ecological Stoichiometry in Vegetable Fields and Orchard in the Coastal Area of Fuzhou].

The soil ecological stoichiometric characteristics of different agricultural land use types have a certain indicator function for characterizing the level of soil nutrient supply and are of great significance to the management of nutrient resources in farmland ecosystems. In order to reveal the soil carbon (C), nitrogen (N), and phosphorus (P) contents and their ecological stoichiometric characteristics in different vegetable fields and orchard agricultural land use types, this study took vegetable fields (taro field and jicama field) and orchards (citrus tree orchard, watermelon field, and pear tree orchard) as the research objects in the coastal area of Fuzhou City. The contents of soil C, N, and P and their ecological stoichiometric characteristics in different vegetable fields and orchard agricultural land uses were measured and analyzed. The soil C and N contents were in the order of orchard>vegetable field (P<0.05). The C content in the citrus tree orchard was the highest (4.44 g·kg-1), and the N content in the watermelon field was the highest (1.46 g·kg-1). The soil P content was vegetable field>orchard (P<0.05), and the jicama field had the highest P content (0.19 g·kg-1). The soil carbon and nitrogen ratio (C/N), carbon and phosphorus ratio (C/P), and nitrogen and phosphorus ratio (N/P) were orchard>vegetable field (P<0.05). Among them, the citrus tree orchard had the highest C/N (7.40) and C/P (61.43), and the watermelon field had the highest N/P (10.27). Soil N content was significantly and negatively correlated with bulk density and conductivity (r=-0.49, r=-0.28, P<0.05), and there was a significant and positive correlation with soil water content (r=0.61, P<0.05). C/P and C/N were significantly and positively correlated with SOM (r=0.71, r=0.64, P<0.01). In the process of crop planting and management in the coastal area of Fuzhou City, it is necessary to reasonably add nitrogen fertilizer to compensate for the N limitation, and slow-release nitrogen fertilizer is better for promoting the sustainable supply of nitrogen nutrients in the growth and development of crops.

A mutated CRYGD associated with congenital coralliform cataracts in two Chinese pedigrees.

AIM: To investigate the causal gene mutation and clinical characteristics for two Chinese families with autosomal dominant congenital coralliform cataract. METHODS: Two Chinese pedigrees with congenital cataract were investigated. Routine ophthalmic examinations were performed on all patients and non-affected family members. Peripheral blood samples were collected, and the genomic DNAs were extracted. The coding regions of proband's DNAs were analyzed with cataract gene panel. The identified mutation was amplified by polymerase chain reaction, and automated sequencing was performed in other members of two families to verify whether the mutated gene was co-segregated with the disease. RESULTS: Congenital coralliform cataract was inherited in an autosomal dominant mode in both pedigrees. For each family, more than half of the family members were affected. All patients presented with severe visual impairment after birth as a result of bilateral symmetric coralliform lens opacification. An exact the same defect in the same gene, a heterozygous mutation of c.70C>A (p. P24T) in exon 2 of γD-crystallin gene, was detected in both probands from each family. Sanger sequencing analysis demonstrated that the mutated CRYGD was co-segregated in these two families. CONCLUSION: A c.70C>A (p. P24T) variant in CRYGD gene was reconfirmed to be the causal gene in two Chinese pedigrees. It is known that mutated CRYGD caused most of the congenital coralliform cataracts, suggesting that the CRYGD gene is associated with coralliform congenital cataract.

Evaluation and application of donors with primary central nervous system tumors.

BACKGROUND: This study aimed to explore the safety of donors with primary central nervous system tumors for kidney and liver transplantations. METHODOLOGY: Clinical data of 29 donors with primary CNS tumors in January 2007 to December 2017, as well as the follow-up data of 16 liver transplant recipients and 46 kidney transplant recipients, were analyzed. According to the risk factors, the high-risk group was classified as Group 1, the low-risk factors were classified as Group 2, and the unknown risk group was classified as Group 3. The incidence of donor-transmitted CNS tumors was calculated and compared. RESULTS: The duration from the diagnosis of 29 donors to donation was 5.67 ± 6.36 months. None of the liver and kidney transplant recipients who were followed up had tumor metastasis. Although the mean survival time of Group 1 was lower than that of Group 2 and Group 3, the Kaplan-Meier curve showed no significant difference in survival time. CONCLUSION: No obvious difference was observed between high-risk and low-risk and unknown risk CNS tumors in terms of the survival rate of transplants and tumor metastasis rate. High-risk CNS tumor donors can be used with the informed consent of recipients after a full evaluation.

An analytical model for the growth and migration of a transverse dune.

The growth and migration speed formulae for a 2-d transverse dune are derived under the assumptions of shape similarity, the near surface airflow independent of height, and the 100% sand trapping efficiency of lee face during dune evolution. Although very simple, this analytical model can quantificationally reflect the field investigations of barchan migrations and the chronological data of mega-dune growth.

Risk of glomerular filtration rate decline in patients with hypertrophic cardiomyopathy and obstructive sleep apnoea.

Sleep apnoea is associated with chronic kidney diseases. A high obstructive sleep apnoea (OSA) prevalence is shown in patients with hypertrophic cardiomyopathy (HCM). Whether the presence of OSA would affect the renal function of patients with HCM is unknown. Forty-five consecutive patients with HCM were divided into the HCM OSA- and OSA+ groups. Forty-three patients with OSA without HCM were recruited as controls. Clinical indices, including estimated glomerular filtration rate (eGFR) and urine 8-hydroxy-2-deoxyguanosine (8-OHdG), were measured. The eGFR was significantly lower in the HCM OSA+ group than in the HCM OSA- (P < 0.05) and OSA (P < 0.001) groups. Multivariate linear regression analysis identified that the apnoea-hypopnoea index was independently associated with eGFR in all patients with HCM (ß = -1.329, 95% confidence interval: -1.942, -0.717, P < 0.001). The urine 8-OHdG level, an oxidative stress marker, was significantly higher in the HCM OSA+ group than in the HCM OSA- (P < 0.001) and OSA (P < 0.001) groups and significantly correlated with the AHI (r = 0.467, P = 0.003) and eGFR (r = -0.457, P = 0.004) in all patients with HCM. Our study suggests a risk of eGFR decline in patients with HCM and OSA.

A novel mutation in fibrillin-1 gene identified in a Chinese family with marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant inheritary disorder of the connective tissue. We report clinical features of a Chinese family with MFS and identify mutations in fibrillin-1 gene (FBN1). In this study, all three members of this family underwent complete ophthalmologic examinations. Two of the three members were diagnosed with MFS. Molecular genetic analysis was performed on the three members. All coding exons of FBN1 were amplified by polymerase chain reaction (PCR). The amplified products were sequenced and compared with a reference sequence in the database. Possible structural and functional changes of the protein induced by amino acids variance were predicted by bioinformatic analysis. A novel heterozygous mutation c.4504 T>A (p.C1502S) in exon 36 was identified in the two affected members, but not in the unaffected member. To our knowledge, this FBN1 mutation has not been reported beforein MFS or other patients.

Single nucleotide polymorphism of MYOC affected the severity of primary open angle glaucoma.

AIM: To detect the mutations in two candidate genes, myocilin (MYOC) and cytochrome P450 1B1 (CYP1B1), in a Chinese family with primary open angle glaucoma (POAG). METHODS: The family was composed of three members, the parents and a daughter. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 genes were screened for sequence alterations by polymerase chain reaction (PCR) and direct DNA sequencing. RESULTS: The mother was the proband, she was diagnosed as POAG in both eyes. Her daughter was diagnosed as juvenile-onset POAG. The father was asymptomatic. One MYOC heterozygous mutation c.1150 G>A (D384N) in exon 3 was identified in the mother, another MYOC heterozygous variation c.1058 C>T (T353I) in exon 3 was identified in the father, and the daughter inherited both of the variations. Meanwhile, three single nucleotide polymorphisms (SNPs) in CYP1B1 gene were found in the family. CONCLUSION: The D384N mutation of MYOC has been reported as one of disease-causing mutations in POAG, whereas T353I variation of MYOC was thought as a high risk factor for POAG. The two variations of MYOC were first reported in one juvenile-onset POAG patient who presented with more severe clinical manifestations, suggesting that T353I polymorphism of MYOC may be associated with the severity of POAG.

[Research progress of the side effect of the prostaglandin drugs].

Glaucoma is one of the leading causes of blindness , second to cataract. Lowering intraocular pressure is the most effective therapeutic means for this disease, with prostaglandin analogs as the first-line medication. These drugs are efficacious and safe, with well-tolerated local adverse effects. They are also generally regarded to have almost no systemic untoward effect. Nonetheless, with the continuous rise in their popularity, systemic adverse effects caused by topical ocular use of prostaglandins have attracted increasing attention. These adverse effects include digestive, respiratory, and cardiovascular systems, as well as skin and hair. Although the incidence of these adverse effects is low, physicians need to be aware of their occurrence. Research progress of the prostacyclin class lOP-lowering medication side effects are reviewed.

Studies of a pedigree with limbal dermoid cyst.

AIM: To study clinical features and gene mutations within the paired-like homeodomain transcription factor 2 (PITX2) gene in a pedigree of bilateral limbal dermoids. METHODS: Complete eye examinations have been performed on each individual of the family. Exons of paired-like homeodomain transcription factor 2 (PITX2) were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: We described the phenotype, clinic findings in a family with two affected members. The masses of the proband's eyes were excised surgically demonstrating a dermoid cyst by histopathological examination. No mutation was detected in the gene PITX2 in this pedigree. CONCLUSION: A family of limbal dermoid cyst was reported. In addition, no pathogenic sequence variations were found in PITX2, indicating that this phenotype in this family is a distinctive entity.

Experimental Tibetan monkey domestication and its application for intraocular pressure measurement.

AIM: To train Tibetan monkey (Macaca thibetana) for intraocular pressure (IOP) measurement in conscious state and obtain normal IOP in conscious Tibetan Macaque. METHODS: The training was based on award-conditioned behavior. Food stimulation and human-animal interaction were used in this training. RESULTS: Trained Tibetan monkeys calmly accepted IOP measurement by the TonoVet® rebound tonometer without sedation or anesthesia and their IOP values were similar to other primates. CONCLUSION: Human-cultivated Thibetan monkeys are tamable, and can be used for biomedical research such as ophthalmic research without anesthesia.

Effects of HepII domain peptides V of fibronectin on corneal permeability, endothelial cells, intraocular pressure and morphology of trabecular meshwork in rats.

BACKGROUND: Trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow in the eye. This study aimed to evaluate the role of HepII domain peptides V on corneal permeability, corneal endothelial cells, intraocular pressure (IOP) and morphology of trabecular meshwork in rats. METHODS: The IOP of rat eyes was measured before and 3, 5, 7 and 8 hours after topical delivery of HepII domain peptides V through intracameral injections. The peptide's concentration in aqueous humor was assessed by high performance liquid chromatography (HPLC). The shape and density of endothelial cells were observed by laser confocal microscopy 8 hours, 3 and 14 days after intracameral injections of HepII domain peptides V. The morphological changes in TM of rat eyes were assessed by transmission electron microscopy (TEM). RESULTS: Intracameral injection of HepII domain peptides V significantly (P < 0.001) decreased IOP by (5.71 ± 2.10) mmHg in rats at 5 hours after injection. There were no obvious changes of the shape and the density of corneal endothelial cells. In addition, morphological changes in the TM of rats were observed including the expansion of intercellular spaces in the juxtacanalicular meshwork, removal of extracellular material, cellular relaxation, and cytoskeleton reorganization. CONCLUSIONS: HepII domain peptides V could not penetrate cornea and was safe to corneal endothelial cells. HepII domain peptides V could significantly decrease IOP in rat probably by disorganizing actin cytoskeleton and cell-junction in the TM.

Arg124Cys mutation of the TGFBI gene in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

AIM: To analyze mutations in transforming growth factor beta-induced (TGFBI) gene in a Chinese pedigree with Reis-Bücklers corneal dystrophy (RBCD, also known as GCD3). METHODS: In a five-generation Chinese family, eight members were identified with RBCD and the rest were unaffected. All members of the family underwent complete ophthalmologic examinations. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all the affected members of the pedigree, but not in the unaffected members. CONCLUSION: R124C which was a known mutation for lattice corneal dystrophy type I, segregated with the RBCD in this pedigree. This elucidated the correlation between genotype and phenotype in a Chinese family of RBCD.