Unpredicted protective function of Fc-mediated inhibitory antibodies for HIV and SARS-CoV-2 vaccines.

Developing effective vaccines is necessary in combating new virus pandemics. For HIV and SARS-CoV-2, the induction of neutralizing antibodies (NAb) is important for vaccine protection; however, the exact mechanisms underlying protection require further study. Recent data emphasize that even Abs that do not exhibit neutralizing activity may contribute to immune defense. Abs exhibiting this function may counter virus mutations, which are acquired to escape from NAbs, and therefore, broaden the protective Ab response induced by vaccination. However, the steps leading to Ab Fc-mediated inhibition are complex. How can these functions be measured in vitro? What inhibitory assay is the most physiologically relevant at mimicking effective in vivo protection? This review provides a comprehensive update on the current knowledge gaps on the Ab Fc-mediated functions involved in HIV and SARS-CoV-2 protection. Understanding the inhibitory effects of these Abs is vital for designing the next generation of protective HIV and SARS-CoV-2 vaccines.

p-Synephrine Indicates Internal Maturity of Citrus grandis (L.) Osbeck cv. Mato Peiyu-Reclaiming Functional Constituents from Nonedible Parts.

The processing of Citrus grandis Osbeck cv. Mato Peiyu (CGMP) fruits generates a considerable amount of waste, mainly the flavedo, albedo, and segment membrane; the generated waste yields severe environmental and economic challenges. In this study, we tried to reclaim some functional chemicals from the waste. Our data indicated that the essential oil content in the flavedo was 0.76-1.34%, with the major component being monoterpenes (93.75% in August, declining to 85.56% in November, including mainly limonene (87.08% to 81.12%) and others such as ß-myrcene). p-Synephrine (mg/100 g dry weight) declined accordingly (flavedo, 10.40 to 2.00; albedo, 1.80 to 0.25; segment membrane, 0.3 in August, 0.2 in September, and none since October). Polyphenols (in µg/g) included gallic acid (70.32-110.25, 99.27-252.89, and 105.78-187.36, respectively); protocatechuic acid (65.32-204.94, 26.35-72.35, and 214.98-302.65, respectively), p-coumaric acid (30.63-169.13, 4.32-17.00, and 6.68-34.32, respectively), ferulic acid (12.36-39.36, 1.21-10.25, and 17.07-39.63, respectively), and chlorogenic acid (59.19-199.36, 33.08-108.57, and 65.32-150.14, respectively). Flavonoids (in µg/g) included naringin (flavedo, 89.32-283.19), quercetin (181.05-248.51), nobiletin (259.75-563.7), hesperidin, and diosmin. The phytosterol content (mg/100 g) was 12.50-44.00 in the flavedo. The total dietary fiber in the segment membrane was 57 g/100 g. The antioxidant activity against the DPPH• and ABTS+• free radicals was moderately high. In conclusion, the waste of CGMP fruits is worth reclaiming for essential oil, p-synephrine, polyphenolics, and dietary fiber. Notably, p-synephrine content (flavedo: <8 mg/100 g dry weight, albedo: <2.0, or segment membrane: <0.4 mg) can serve as a marker of the internal maturation of CGMP fruits.

Fc receptors and the diversity of antibody responses to HIV infection and vaccination.

The development of an effective vaccine against HIV is desperately needed. The successive failures of HIV vaccine efficacy trials in recent decades have shown the difficulty of inducing an appropriate protective immune response to fight HIV. Different correlates of antibody parameters associated with a decreased risk of HIV-1 acquisition have been identified. However, these parameters are difficult to reproduce and improve, possibly because they have an intricate and combined action. Here, we describe the numerous antibody (Ab) functions associated with HIV-1 protection and report the interrelated parameters regulating their complex functions. Indeed, besides neutralizing and Fc-mediated activity, additional factors such as Ab type, concentration and kinetics of induction, and Fc-receptor expression and binding capacity also influence the protective effect conferred by Abs. As these parameters were described to be associated with ethnicity, age and sex, these additional factors must be considered for the development of an effective immune response. Therefore, future vaccine designs need to consider these multifaceted Ab functions together with the demographic attributes of the patient populations.

A dual-signal sensing strategy based on ratiometric fluorescence and colorimetry for determination of Cu2+ and glyphosate.

Herein, a dual-signal sensing strategy based on ratiometric fluorescence and colorimetry for Cu2+ and glyphosate determination was constructed. Fluorescence silicon nanoparticles (SiNPs) were prepared by hydrothermal reaction, which has maximum fluorescence intensity under the excitation of 355 nm. o-Phenylenediamine (OPD) was oxidized through Cu2+ to generate 2,3-diaminophenazine (oxOPD). The obtained oxOPD showed a strong absorption peak at 417 nm and quenched the fluorescence of SiNPs at 446 nm due to fluorescence resonance energy transfer (FRET). Meanwhile, oxOPD produced a new fluorescence emission at 556 nm forming a ratiometric state. With increasing Cu2+, the original solution changed from colorless to yellow. When glyphosate was present, the interaction between Cu2+ and the functional groups of glyphosate could reduce the oxidation of oxOPD, resulting in the enhancement of fluorescence at 446 nm and the decrease of fluorescence at 556 nm. Furthermore, the addition of glyphosate changed yellow solution to colorless. Under the optimal conditions of OPD (1 mM), 20 mM Tris-HCl buffer (pH 7.5), and incubation time (4 h), the ratiometric fluorescence sensor had good selectivity and showed a wide linear range of 0.025-20 µM with the LOD of 0.008 µM for Cu2+ and 0.15-1.5 µg/mL with the LOD of 0.003 µg/mL for glyphosate, respectively. Besides, it is worth mentioning that this developed sensing system showed good performance in real samples, providing a simple and reliable dual-signal detection strategy.

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion.

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

Volatile Variation of Theobroma cacao Malvaceae L. Beans Cultivated in Taiwan Affected by Processing via Fermentation and Roasting.

After being harvested, cacao beans are usually subjected to very complex processes in order to improve their chemical and physical characteristics, like tastefulness with chocolate characteristic flavors. The traditional process consists of three major processing stages: fermentation, drying, and roasting, while most of the fermentation is carried out by an on-farm in-box process. In Taiwan, we have two major cocoa beans, the red and the yellow. We proposed that the major factor affecting the variation in tastes and colors in the finished cocoa might be the difference between cultivars. To uncover this, we examined the effect of the three major processes including fermentation, drying and roasting on these two cocoa beans. Results indicated that the two cultivars really behaved differently (despite before or after processing with fermentation, drying, and roasting) with respect to the patterns of fatty acids (palmitic, stearic, oleic, and arachidonic); triacylglycerols:1,2,3-trioleoyl-glycerol (OOO); 1-stearoyl-2,3-oleoyl-glycerol (SOO); 1-stearoyl-sn-2-oleoyl-3-arachidoyl- glycerol (SOA); 1,3-distearyol-sn-2-oleoyl-glycerol (SOS); organic acids (citric, tartaric, acetic, and malic); soluble sugars (glucose and fructose); amino acids; total phenolics; total flavonoids; and volatiles. Our findings suggest that to choose specific processing conditions for each specific cocoa genotype is the crucial point of processing cocoa with consistent taste and color.

Analysis of Volatile Compounds from Different Parts of Houttuynia cordata Thunb.

Houttuynia cordata Thunb. is a medicinal and edible plant that has been commonly used in traditional Chinese medicine since ancient times. This study used headspace solid-phase microextraction (HS-SPME) and direct injection, combined with gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), to identify the volatile compounds in H. cordata. Extraction from different parts of the plant using different extraction techniques for the identification of volatile compounds were determined. A total of 93 volatile components were analyzed in the leaves, stems, rhizomes, and whole plant samples of H. cordata. The leaves contained more (Z)-3-hexenal, ß-myrcene, (Z)-ß-ocimene, and (4E,6E)-allo-ocimene; the stems contained more geranyl acetate and nerolidol; and rhizomes contained more α-pinene, ß-pinene, limonene, 2-undecanone, and decanoyl acetaldehyde. Among them, the essential oil extracted by HS-SPME could produce more monoterpenes, while direct injection could obtain higher contents of aliphatic ketones, terpene esters, sesquiterpenes, and was more conducive to the extraction of 2-undecanone and decanoyl acetaldehyde.

Molecular Action Mechanism of Coixol from Soft-Shelled Adlay on Tyrosinase: The Future of Cosmetics.

Coix lacryma-jobi var. ma-yuen L. Gramineae is widely cultivated in Taiwan. Literature regarding the molecular action mechanism of coixol on tyrosinase and the application of coicis seed extracts to the processing of facial masks is still lacking. Solvent extractability analysis revealed that most of the polyphenolics in coicis seeds were water soluble (3.17 ± 0.12 to 3.63 ± 0.07 µg/mLGAE). In contrast, the methanolic extract contained the most flavonoids (0.06 ± 0.00~0.26 ± 0.03 µg/mL QE) and coixol (11.43 ± 0.13~12.83 ± 0.14 µg/mL), showing potent antioxidant capability. Additionally, the contents of coixenolide (176.77 ± 5.91 to 238.60 ± 0.21 µg/g), phytosterol (52.45 ± 2.05 to 58.23 ± 1.14 mg/g), and polysaccharides (3.42 ± 0.10 to 4.41 ± 0.10 mg/g) were rather high. The aqueous extract (10 µg/mL) and the ethanolic extract (1 mg/mL) showed no cytotoxicity to B16F10 melanocytes. More attractively, the ethanolic extract at 1 mg/mL caused 48.4% inhibition of tyrosinase activity in B16F10 melanocytes, and 50.7% on human tyrosinase (hTyr) fragment 369-377. Conclusively, the coicis seed extracts containing abundant nutraceuticals with promising anti-hTyr activity and moisturizing capability can serve as good ingredients for facial mask processing.

Diversity and abundance of diazotrophic communities of seagrass Halophila ovalis based on genomic and transcript level in Daya Bay, South China Sea.

Seagrass ecosystems are among the most productive marine ecosystems, and diazotrophic communities play a crucial role in sustaining the productivity and stability of such ecosystems by introducing fixed nitrogen. However, information concerning both total and active diazotrophic groups existing in different compartments of seagrass is lacking. This study comprehensively investigated the diversity, structure, and abundance of diazotrophic communities in different parts of the seagrass Halophila ovalis at the DNA and RNA level from clone libraries and real-time quantitative PCR. Our results indicated that nearly one-third of existing nitrogen-fixing bacteria were active, and their abundance might be controlled by nitrogen to phosphorus ratio (N:P). Deltaproteobacteria and Gammaproteobacteria were dominant groups among the total and active diazotrophic communities in all samples. These two groups accounted for 82.21% and 70.96% at the DNA and RNA levels, respectively. The genus Pseudomonas and sulfate-reducing bacteria (genera: Desulfosarcina, Desulfobulbus, Desulfocapsa, and Desulfopila) constituted the significant fraction of nitrogen-fixing bacteria in the seagrass ecosystem, playing an additional role in denitrification and sulfate reduction, respectively. Moreover, the abundance of the nitrogenase gene, nifH, was highest in seawater and lowest in rhizosphere sediments from all samples. This study highlighted the role of diazotropic communities in the subtropical seagrass ecosystem.

Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (EBA-175) gene in Equatorial Guinea.

BACKGROUND: Plasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms and dimorphism have prevented to development of effective vaccines based on this gene. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko Island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175. METHODS: The allelic dimorphism of PfEBA-175 region II of 297 bloods samples from Equatorial Guinea in 2018 and 2019 were investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program. RESULTS: Both Bioko Island and Bata district populations, the frequency of the F-fragment was higher than that of the C-fragment of PfEBA-175 gene. The PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and - 0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (FST > 0.15, P < 0.05). A total of 310 global isolates clustered in 92 haplotypes, and only one cluster contained isolates from three continents. The mutations A34T, K109E, D278Y, K301N, L305V and D329N were predicted as probably damaging. CONCLUSIONS: This study demonstrated that the dimorphism of F-fragment PfEBA-175 was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar another region isolates. And the levels of recombination events suggested that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen.

Genetic diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium falciparum on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described. METHODS: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools. RESULTS: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure. CONCLUSIONS: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Fundamentals of Optical Imaging.

Optical imaging, which possesses noninvasive and high-resolution features for biomedical imaging, has been used to study various biological samples, from in vitro cells, ex vivo tissue, to in vivo imaging of living organism. Furthermore, optical imaging also covers a very wide scope of spatial scale, from submicron sized organelles to macro-scale live biological samples, enabling it a powerful tool for biomedical studies. Before introducing these superior optical imaging methods to researchers, first of all, it is necessary to present the basic concept of light-matter interactions such as absorption, scattering, and fluorescence, which can be used as the imaging contrast and also affect the imaging quality. And then the working mechanism of various imaging modalities including fluorescence microscopy, confocal microscopy, multiphoton microscopy, super-resolution microscopy, optical coherence tomography (OCT), diffuse optical tomography (DOT), etc. will be presented. Meanwhile, the main features and typical bioimaging applications of these optical imaging technologies are discussed. Finally, the perspective of future optical imaging methods is presented. The aim of this chapter is to introduce the background and principle of optical imaging for grasping the mechanism of advanced optical imaging modalities introduced in the following chapters.

The diversity of alkane-degrading bacterial communities in seagrass ecosystem of the South China Sea.

Seagrass meadows are one of the most important marine ecosystems. Alkanes are the common hydrocarbon contaminants that can affect seagrass growth. In this study, a large spatial-scale investigation has been carried out on the alkane-degrading bacterial community structure in the rhizosphere and non-rhizosphere sediments of two seagrass species (Thalassia hemprichii and Halophila ovalis). AlkB gene was employed as a biomarker gene to study the alkane-degrading bacterial community structure. The results showed that the alpha diversity of the alkane-degrading bacterial community in T. hemprichii non-rhizosphere sediments was higher than that of its rhizosphere sediments. However, the alpha diversity of the alkane-degrading bacterial community in H. ovalis rhizosphere sediments was higher than that of its non-rhizosphere sediments in the open sea, but the result was contrast in the coast area. In addition, the alpha diversity of alkane-degrading bacterial communities in the coast area was higher than that of far away from the coast in the T. hemprichii rhizosphere and non-rhizosphere sediments. The phylogenetic analysis result revealed that the alkB sequences from the seagrass ecosystem were mainly affiliated with the class Alphaproteobacteria, and had the two novel lineages. Genus Agrobacterium was the most predominant alkane-degrading bacteria. These results contributed to disclose the geographical distribution pattern of alkane-degrading bacteria in the seagrass ecosystem of the South China Sea.

The distribution characteristics of ß-propeller phytase genes in rhizosphere sediment provide insight into species specialty from phytic mineralization in subtropical and tropical seagrass ecosystems.

Seagrass meadows have seriously deteriorated in recent years. Seagrass associated phytate-mineralizing rhizobacteria potentially have functions related to seagrass nutrition, health and sustainable growth. The ß-propeller phytases (BPPs) are the only phytase family in aquatic environments, but there are few studies on the BPP community structure of seagrass. In this study, clone libraries and quantitative PCR (qPCR) assays were used to compare the diversity and abundances of the BPP communities of Halodule endl, Halophila ovalis and Thalassia hemprichii in Xisha and Sanya, and to investigate the distribution characteristics of BPP genes in the rhizosphere sediment, which provedes insight into species specialty from phytic mineralization in subtropical and tropical seagrass ecosystems. The highest diversity of BPP genes was found for Thalassia hemprichii in Sanya Bay. Thalassia hemprichii in Sanya had higher abundances of BPPs, which were linked to Gammaproteobacteria. The BPP community diversity and OTUs of Thalassia hemprichii in Sanya were much higher than those of Thalassia hemprichii on Yongxing Island and Stone Island. The seagrass BPP communities had higher diversity and evenness from sampling sites with more human activity. The qPCR results showed that the abundance of phytate acid-degradating bacteria was approximately three times larger in Thalassia hemprichii rhizosphere sediment samples than in Halodule endl and Halophila ovalis rhizosphere sediment samples. This study highlighted that the diversity and abundances of bacteria genetically encoding BPP in the rhizosphere of Thalassia hemprichii were clearly higher than those of Halodule endl and Halophila ovalis. Further study of microbial phosphorus cycling will provide new insights into seagrass meadow ecosystems.

Genetic polymorphism of Plasmodium falciparum circumsporozoite protein on Bioko Island, Equatorial Guinea and global comparative analysis.

BACKGROUND: Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population. METHODS: From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI. RESULTS: In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p < 0.05). The comparative analysis of Bioko and global pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p < 0.05). The global pfcsp C-terminal sequences were clustered into 138 different haplotypes (H_1 to H_138). Only 3.35% of sequences matched 3D7 strain haplotype (H_1). CONCLUSIONS: The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.

Curcumin Suppresses Apoptosis and Inflammation in Hypoxia/Reperfusion-Exposed Neurons via Wnt Signaling Pathway.

BACKGROUND Cerebral ischemia/reperfusion (I/R) injury contributes to mortality and morbidity in preterm infants. Curcumin has been shown to exert neuro-protective effects in the central nervous system (CNS). The aim of this study was to investigate the neuro-protective activity of curcumin and the possible underlying molecular mechanisms. MATERIAL AND METHODS A hypoxia/reoxygenation (H/R) protocol was used to simulate I/R injury in vitro. Isolated neonatal neurons were pre-treated with curcumin at serially diluted concentrations and exposed to H/R injury. Cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. Contents of TNFa and IL6 in supernatant of cell culture medium were detected by ELISA. Protein expression, phosphorylation, and nuclear translocation levels were studied by Western blotting. RESULTS H/R reduced cell viability and increased apoptosis of neurons. H/R significantly increased Wnt5a expression, JNK1 phosphorylation, and NF-kappaB nuclear translocation. Moreover, expression levels of cleaved caspase3, TNFalpha, and IL6 were elevated in H/R-exposed neurons. Curcumin pre-treatment significantly increased cell viability and inhibited apoptosis of neurons exposed to H/R, in a concentration-dependent manner. Moreover, curcumin pre-treatment significantly decreased expression levels of Wnt5a, IL6, TNFalpha, and phosphorylation level of JNK1, as well as the nuclear translocation level of NF-kappaB in H/R-exposed neurons, in a concentration-dependent manner. CONCLUSIONS Curcumin exerted neuro-protective effects against H/R-induced neuron apoptosis and inflammation by inhibiting activation of the Wnt/JNK1 signaling pathway.

P,N Codoped carbon dots as an efficient "off-on" fluorescent probe for lipoic acid detection and its cellular dual-color imaging.

A facile single hydrothermal method was developed to synthetize P,N codoped carbon dots (P,N/CDs), which show strong and stable fluorescence, good water solubility, low toxicity and good biocompatibility. Hence, a novel and efficient "off-on" P,N/CDs fluorescent probe was developed for the highly sensitive detection of lipoic acid (LA) for the first time. The fluorescence of the P,N/CDs was quenched by Cu2+ forming a P,N/CDs-Cu2+ complex, which acted as the "off" process, but Cu2+ could be removed by LA, due to stronger chelating between LA and Cu2+, forming a more stable complex, which recovered the fluorescence of the P,N/CDs, in order to achieve the "on" process. Under optimal conditions, the concentration of LA and the increased fluorescence intensity of the P,N/CDs-Cu2+ complex displayed a good linear relationship within the range of 0.05-28 µM, with a detection limit (S/N = 3) of 0.02 µM. The established "off-on" fluorescent probe was successfully applied to the analysis of LA in urine samples. The average recoveries were in the range of 98.3-101.5%, with a relative standard deviations of less than 3.1%. In addition, the P,N/CDs were also successfully applied to cellular dual-color imaging of live T24 cells. The results show that the P,N/CDs have great application potential in clinical diagnosis, bioassay and bioimaging. Graphical abstract.

[Three novel splicing mutations at 5' terminal of DMD gene corresponding to different phenotypes].

OBJECTIVE: To study the correlation of splicing mutations at the 5' end of the DMD gene with their phenotypes. METHODS: DMD gene mutations were analyzed using Multiplex Ligation Probe Amplification (MLPA) and Sanger sequencing. Co-segregation analysis was performed for the pedigrees of the probands. Influence of mutations on protein function was predicted by bioinformatic analysis. RESULTS: Three novel splicing mutations were identified in three patients with different phenotypes. Patient 1 carried a c.31+3insT mutation and presented primarily with dilated cardiomyopathy (XLDC). There was no clinical signs of skeletal myopathy. Bioinformatic analysis predicted that the mutation may inactivate the splicing donor of intron 1 and lead to premature termination of protein translation. Patient 2 carried a c.264_264+4delTGTAA mutation, which led to loss of splicing donor site for intron 4, and manifested Becker muscular dystrophy (BMD). The mutation was predicted to result in skipping of exon 4. The defective protein may still retain most of its function. Patient 3 had Duchenne muscular dystrophy (DMD) and carried a c.832-3C>T mutation which was predicted to decrease the activity of splicing acceptor of intron 8, resulting in usage of alternative acceptor site or retain of intron 8. All related transcripts may cause premature termination of protein translation and complete loss of protein function. The three mutations were all inherited from the mothers of the patients. CONCLUSION: Three novel splicing mutations were discovered at the 5' end of DMD gene in three patients with different disease phenotypes. Our study may facilitate understanding of the influence of splicing mutations at the 5' end of the DMD gene on dystrophin function and the correlation between genotypes and phenotypes.

WNT10A variants are associated with non-syndromic tooth agenesis in the general population.

Tooth agenesis is the most common developmental dental anomaly. Absence of one or two permanent teeth is found in the majority of affected subjects. Very few patients suffer severe tooth agenesis. Recent studies revealed that WNT10A gene mutations caused syndromic and isolated severe tooth agenesis. In this study, to determine the contribution of WNT10A variants in different severities of tooth agenesis, we investigated the association between WNT10A variants and non-syndromic tooth agenesis in a Chinese population consisting of 505 tooth agenesis patients and 451 normal controls. Twenty-three novel non-synonymous variants were identified. WNT10A variants were detected in 15.8 % (75/474) of patients with 1-3 missing teeth and 51.6 % (16/31) of patients with 4 or more missing teeth. As compared with a frequency of 3.1 % in individuals with full dentition, variant allele frequencies were significantly elevated in both groups with tooth agenesis (p values of 1.00 × 10(-6) and 3.89 × 10(-23), respectively). Our findings showed that WNT10A variants were associated with non-syndromic tooth agenesis from mild to severe tooth agenesis, and the more severe tooth agenesis, the stronger association. Biallelic genotypes of WNT10A variants may have a pathogenic effect on tooth development. Presence of a single variant allele would be predisposing for causation with low penetrance. Together with WNT10A variant, there should be other genetic or environmental factors leading to biallelic variant-related variable clinical manifestations and single allele variant-related low penetrance. The frequent missing tooth positions in the WNT10A-related cases were consistent with that in the general population, suggesting WNT10A plays a critically important role in the etiology of general tooth agenesis.

Headspace solid-phase microextraction analysis of volatile components in Phalaenopsis Nobby's Pacific Sunset.

Phalaenopsis is the most important economic crop in the Orchidaceae family. There are currently numerous beautiful and colorful Phalaenopsis flowers, but only a few species of Phalaenopsis have an aroma. This study reports the analysis volatile components present in P. Nobby's Pacific Sunset by solid-phase microextraction (SPME) coupled with gas chromatography (GC) and gas chromatography/mass spectrometry (GC-MS). The results show that the optimal extraction conditions were obtained by using a DVB/CAR/PDMS fiber. A total of 31 compounds were identified, with the major compounds being geraniol, linalool and α-farnesene. P. Nobby's Pacific Sunset had the highest odor concentration from 09:00 to 13:00 on the eighth day of storage. It was also found that in P. Nobby's Pacific Sunset orchids the dorsal sepals and petals had the highest odor concentrations, whereas the column had the lowest.