Characterization of Chinese Unifloral Honeys Based on Proline and Phenolic Content as Markers of Botanical Origin, Using Multivariate Analysis.

The phenolic and proline content were determined in honey samples of different floral origins (rapeseed, sunflower, buckwheat and Codonopsis) from five different regions of China. The phenolic and proline profile of these samples were used to construct a statistical model to distinguish honeys from different floral origins. Significant differences were identified among the studied honey samples from multivariate chemometric methods. The proline content varied among the four types of honeys, with the values decreasing in the order: buckwheat > Codonopsis > sunflower > rapeseed. Rapeseed honeys contained a high level of benzoic acid, while rutin, p-coumaric acid, p-hydroxybenzoic acid were present at relatively high levels in buckwheat honeys. Principal component analysis (PCA) revealed that rapeseed honey could be distinguished from the other three unifloral honeys, and benzoic acid, proline and kaempferol could serve as potential floral markers. Using 18 phenolic compounds and proline the honey samples were satisfactorily classified according to floral origin at 94% correct prediction by linear discriminant analysis (LDA). The results indicated that phenolic compounds and proline were useful for the identification of the floral origin of the four type honeys.

Using the combined analysis of transcripts and metabolites to propose key genes for differential terpene accumulation across two regions.

BACKGROUND: Terpenes are of great interest to winemakers because of their extremely low perception thresholds and pleasant floral odors. Even for the same variety, terpene profile can be substantially different for grapevine growing environments. Recently a series of genes required for terpene biosynthesis were biochemically characterized in grape berries. However, the genes that dominate the differential terpene accumulation of grape berries between regions have yet to be identified. METHODS: Free and glycosidically-bound terpenes were identified and quantified using gas chromatography-mass spectrometry (GC-MS) technique. The transcription expression profiling of the genes was obtained by RNA sequencing and part of the results were verified by quantitative real time PCR (QPCR). The gene co-expression networks were constructed with the Cytoscape software v 2.8.2 ( www.cytoscape.org). RESULTS: 'Muscat Blanc a Petits Grains' berries were collected from two wine-producing regions with strikingly different climates, Gaotai (GT) in Gansu Province and Changli (CL) in Hebei Province in China, at four developmental stages for two consecutive years. GC-MS analysis demonstrated that both free and glycosidically bound terpenes accumulated primarily after veraison and that mature grape berries from CL contained significantly higher concentrations of free and glycosidically bound terpenes than berries from GT. Transcriptome analysis revealed that some key genes involved in terpene biosynthesis were markedly up-regulated in the CL region. Particularly in the MEP pathway, the expression of VviHDR (1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase) paralleled with the accumulation of terpenes, which can promote the flow of isopentenyl diphosphate (IPP) into the terpene synthetic pathway. The glycosidically bound monoterpenes accumulated differentially along with maturation in both regions, which is synchronous with the expression of a monoterpene glucosyltransferase gene (VviUGT85A2L4 (VviGT14)). Other genes were also found to be related to the differential accumulation of terpenes and monoterpene glycosides in the grapes between regions. Transcription factors that could regulate terpene synthesis were predicted through gene co-expression network analysis. Additionally, the genes involved in abscisic acid (ABA) and ethylene signal responses were expressed at high levels earlier in GT grapes than in CL grapes. CONCLUSIONS: Differential production of free and glycosidically-bound terpenes in grape berries across GT and CL regions should be related at least to the expression of both VviHDR and VviUGT85A2L4 (VviGT14). Considering the expression patterns of both transcription factors and mature-related genes, we infer that less rainfall and stronger sunshine in the GT region could initiate the earlier expression of ripening-related genes and accelerate the berry maturation, eventually limiting the production of terpene volatiles.

Electrophysiological and microperimetry changes in vitamin A deficiency retinopathy.

PURPOSE: To describe a follow-up case of vitamin A deficiency (VAD)-related retinopathy with macular involvement monitored with electroretinography and microperimetry. METHODS: Flash (ERG), pattern (PERG), and multifocal (mfERG) electroretinography were recorded in parallel with microperimetry before and during 7-month follow-up of vitamin A treatment on a 65-year-old man presented with a 1-year history of night blindness. The patient had undergone ileostomy procedure for Crohn's disease 14 years ago. His best corrected visual acuity was 6/6 in each eye. Fundus examination revealed macular and mid-peripheral yellow-white lesions compatible with drusen. RESULTS: Before treatment, PERG was reduced, and mfERG was reduced and delayed, worse in responses from the central rings. These results revealed evidence of bilateral macular and central cone dysfunction. The flash ERG showed reduced dark-adapted 0.01 ERG (rod response); a-wave of dark-adapted 3.0 and 10.0 ERG was normal but b-wave was reduced (negative configuration); light-adapted 3.0 ERG was slightly reduced. The flash ERG revealed evidence of bilateral generalised retinal dysfunction affecting the rod more than cone system. Microperimetry showed deep reduction in retinal sensitivity. Fixation stability was unstable with eccentric locus. During treatment, ERG and microperimetry demonstrated significant improvements. Fixation stability reached normal values after the third treatment week. CONCLUSIONS: This case illustrates the importance of electrophysiological investigation in early VAD-related retinopathy detection correlated with microperimetry. Our findings indicate a more delayed central cone function recovery than that of generalised rod and cone function. There was a marked correlation between ERG and microperimetric changes.

Visual evoked potential (VEP) and multifocal electroretinogram (mfERG) in ocular syphilitic posterior segment inflammation.

The aim of this study is to correlate multifocal electroretinogram (mfERG) and visual evoked potential (VEP) changes with visual acuity and clinical features in patients with posterior segment inflammation secondary to syphilis. A retrospective interventional case series of 4 patients with visual loss secondary to syphilitic uveitis is reported. The mfERG (P1) showed diminished amplitudes and prolonged latency in 7 affected eyes. Visual acuity rapidly improved 2 weeks after initiation of therapy. OCT demonstrated anatomical recovery at 1 month. In three patients, visual acuity was restored to 6/6 at 6-9 months but mfERG responses remained significantly reduced and delayed for 12-15 months before recovery to normal levels. One patient developed a retinal detachment, but achieved 6/9 vision at 30 months. VEP changes, interpreted in combination with mfERG responses, showed evidence of optic nerve involvement in 6 eyes. Ocular findings, including posterior placoid chorioretinitis, are important diagnostic features of secondary and tertiary syphilis. Visual acuity and clinical recovery occur early with appropriate diagnosis and treatment, and precede full electrophysiological recovery of the outer retina-RPE complex. Ophthalmologists have the opportunity to play a key role in undetected or missed diagnoses of syphilis, and with appropriate treatment the visual prognosis is excellent.

Molecular characterization and expression analysis on two isogenes encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in grapes.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) is an entry enzyme of the shikimate pathway that connects primary carbohydrate metabolism with the biosynthesis of most secondary metabolites in plants. In the present study, two DAHPS cDNAs were cloned from grape berries (Vitis vinifera) and designated as VvDAHPS-1 and VvDAHPS-2. These two cDNA sequences share 75.7% of the identities. Their DNA corresponding to the two isogenes both contain four introns. The deduced proteins from two cDNAs had different NH(4)-terminal regions and putative mature regions sharing sequence, molecular size and pI value similarity. Both of VvDAHPSs had a close evolution relationship with Populus trichocarpa DAHPSs. The prokaryotically-expressed VvDAHPSs both manifested DAHPS catalytic activity and Mn(2+)-activated effects. Analysis by real time-PCR showed that VvDAHPS-1 and VvDAHPS-2 were expressed in all the tested tissues, but their expression patterns accompanying with berry mature varied in the skin, pulp and seeds. The results give new insight into further study on regulatory mechanism of grape phenolics biosynthesis.

Antibody preparation, gene expression and subcellular localization of L-idonate dehydrogenase in grape berry.

L-Idonate dehydrogenase (L-IdnDH) is the only known enzyme of the tartaric acid (TA) biosynthetic pathway in grape berries. Previous studies on this enzyme only focused on the transcriptional expression of this gene. Here, the antibody against L-IdnDH was prepared by cloning the full-length cDNAs of VvIdnDH from grape berries, highly expressing fusion protein in Escherichia coli cells, and then immunizing rabbits. Expression of VvIdnDH at the transcriptional and translational levels in grape berries increased within 3 weeks after full bloom and then decreased, similarly to the trend for TA contents. Almost no VvIdnDH transcript was detected in the mature berries, but VvIdnDH protein was detectable. In the 21-d berries, this enzyme was largely located in the cytoplasm and a few in the cell wall, secondary cell wall, and chloroplast, whereas in the 60 berries, it was distributed mainly in the vacuole and cytoplasm.

Monoterpenyl Glycosyltransferases Differentially Contribute to Production of Monoterpenyl Glycosides in Two Aromatic Vitis vinifera Varieties.

HIGHLIGHTS A similar trend on accumulation of glycosidically bound monoterpenes was observed in both varietiesTwo VvGT7 alleles mutations occurred at key sites in Muscat blanc à PetitVvGT14 exerted a major role in production of monoterpenyl glycosides in both varieties Terpenoids are the major aroma components and generally exist as both free and glycosidically-bound forms, of which nonvolatile glycosides account for a large fraction in grape berries. Our previous study has indicated that differential accumulation of monoterpenyl glycosides in Vitis vinifera "Muscat blanc à Petit" between two regions is closely correlated to monoterpenyl glucosyltransferase (VvGT14, XM_002285734.2) transcript abundance. However, it has not been determined yet whether this correlation also exists in other Vitis vinifera varieties. This study investigated the evolution of free and glycosidically bound monoterpenes in two Vitis vinifera variety "Muscat blanc à Petit" and "Gewurztraminer" under two vintages, and further assessed the relation between the accumulation of bound monoterpenes and two monoterpenyl glycosyltransferase transcript levels. Results showed that free monoterpenes exhibited three evolution patterns in both varieties during berry development of two vintages, whereas glycosidically bound monoterpenes showed a concentration elevation with berry maturation. The Cis-rose oxide and geraniol were major components contributing to the aroma odors of "Gewürztraminer" grapes while linalool was major aroma contributor to the "Muscat blanc à Petit grain" grapes. The accumulation of glycosidically bound monoterpenes in both varieties was accompanied with the high expression of VvGT7 (XM_002276510.2) and VvGT14. Only one allele of VvGT7 was found in the variety "Gewürztraminer" and no mutation was observed in its enzyme active sites. XB-VvGT7-4 and XB-VvGT7-5 were two alleles of VvGT7 detected in "Muscat blanc à Petit grain." The mutation on its enzyme active site inhibited the activity of XB-VvGT7-4, whereas VvGT7-5 exhibited an alteration on enzyme activity due to the insertion mutation at the position 443. Only one VvGT14 allele was found in both varieties, and the VvGT14 allele in both varieties showed the similarity on amino acid sequence. No mutation occurred in active sites of VvGT14 allele. These indicated that VvGT7 and VvGT14 differentially contributed to the production of monoterpenyl glycosides in these Vitis Vinifera varieties.

The Microbial Community Dynamics during the Vitex Honey Ripening Process in the Honeycomb.

The bacterial and fungal communities of vitex honey were surveyed by sequencing the 16S rRNA gene and the internal transcribed spacer region of ribosomal DNA. Vitex honey samples were analyzed at different stage of ripening; the vitex flower was also analyzed, and the effect of the chemical composition in the experimental setup was assessed. The results confirmed the presence of dominant Bacillus spp. as the dominant bacterial in honey, and yeast related genera was the main fungal in the honey, respectively. Lactococcus and Enterococcus were detected for the first time in honey. The proportion of most of the fungal community decreased during the honey ripening process. Multivariate analyses also showed that the fungal community of 5, 10, and 15 days honey samples tended to cluster together and were completely separated from the 1 day honey sample. The change in the fungal community showed a correlation with the variation in the chemical components, such as moisture and phenolic compounds. Together, these results suggest that ripening of honey could change its microbial composition, and decrease the potential risk of microbiology.

Strategy for comparative untargeted metabolomics reveals honey markers of different floral and geographic origins using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry.

Honey discrimination based on floral and geographic origins is limited by the ability to determine reliable markers because developing hypothetical substances in advance considerably limits the throughput of metabolomics studies. Here, we present a novel approach to screen and elucidate honey markers based on comparative untargeted metabolomics using ultrahigh-performance liquid chromatography-hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Orbitrap). To reduce metabolite information losses during sample preparation, the honey samples were dissolved in water and centrifuged to remove insoluble particles prior to UHPLC-Q-Orbitrap analysis in positive and negative electrospray ionization modes. The data were pretreated using background subtraction, chromatographic peak extraction, normalization, transformation and scaling to remove interferences from unwanted biases and variance in the experimental data. The pretreated data were further processed using principal component analysis (PCA) and a three-stage approach (t-test, volcano plot and variable importance in projection (VIP) plot) to ensure marker authenticity. A correlation between the molecular and fragment ions with a mass accuracy of less than 1.0ppm was used to annotate and elucidate the marker structures, and the marker responses in real samples were used to confirm the effectiveness of the honey discrimination. Moreover, we evaluated the data quality using blank and quality control (QC) samples based on PCA clustering, retention times, normalized levels and peak areas. This strategy will help guide standardized, comparative untargeted metabolomics studies of honey and other agro-products from different floral and geographic origins.

[Polymorphism of porcine myostatin gene].

Myostatin(MSTN) gene is expressed specifically in developing and mature skeletal muscle, and the expression products of MSTN gene inhibits muscle growth and differentiation. The polymorphism of the porcine MSTN gene was researched by PCR-SSCP. The SSCP was found in the MSTN gene exon 2 and exon 3. It showed three genotypes (CC, CT, TT) in exon 2 and two genotypes (AG, GG) in exon 3 in Large White pigs. The relationship between polymorphism in exons 2 and 3 and productive performance was analysed. Variance analysis showed that there was no significant relationship between the polymorphism in exon 2 and productive performance. But t test showed that there was a significant relationship between the polymorphism in exon 3 and back fat thickness (P < 0.05), there was also a relationship between the polymorphism in exon 3 and lean meat percentage, but not significant (P > 0.05). The fragments with SSCP polymorphism in exon 2 and 3 were sequenced, the sequencing results showed that there were two single nucleotides mutation, i.e. A-->G at 1008 (exon 3) and G-->T at 480 (exon 2). Two mutations did not change the amino acid but brought an Apa I site in exon 3, and PCR-RFLP molecular marker technique was established with Apa I.

Free and glycosidically bound aroma compounds in cherry (Prunus avium L.).

This paper reports the occurrence of both free and glycosidically bound aroma compounds in three sweet cherry cultivars ('Hongdeng', 'Hongyan' and 'Rainier'), with 97 compounds being identified in the three cultivars. The major free volatile compounds found were hexanal, (E)-2-hexenal, (E)-2-hexen-1-ol, benzyl alcohol and benzaldehyde. The major bound volatile compounds found were benzyl alcohol, geraniol, 2-phenylethanol. Also 4-vinylphenol was found in cherry fruit for the first time, and has a relatively high concentration of the glycosidically-bound form in 'Rainier'. Odour activity values (OAVs) were determined for both free and bound volatiles, with 18 compounds having an OAV above 1. The highest OAVs for three cultivars were (E)-ß-ionone, hexanal, decanal and (E)-2-hexenal with the highest being over 800 for (E)-ß-ionone in 'Honyang'. From these results, it was concluded that the aroma compounds present were similar in all three cultivars, but there was significant variation found in their levels and hence contribution to the aroma of these cultivars.

Three types of ultraviolet irradiation differentially promote expression of shikimate pathway genes and production of anthocyanins in grape berries.

Modulation of flavonoid biosynthesis in grape berries has always aroused great attention among researchers. However, little study has been made on the shikimate pathway that guides photo-assimilate flow into flavonoid metabolism. The present study indicated that the treatments of three ultraviolet (UV) wavelengths differentially up-regulated transcriptional expression of some structural genes in the shikimate pathway and post-chorismate pathway of grape berries and this up-regulation was developmental stage-dependent and not synchronous. Of these genes, VvDAHPS-1 and VvDAHPS-2 encoding the entry enzymes of the shikimate pathway showed most significant UV-response and their transcription was strongly promoted by UV-A stimuli in the 3-week grapes and by UV-B and UV-C in the 7-week and 11-week grapes. The elevation of VvAS expression by UV induction appeared in the 3-week grapes and VvCM-1 was expressed relatively more concomitantly with berry mature. Correspondently, UV-B and UV-C irradiation increased the content of various anthocyanins in the 11-week grapes, but UV-A did not. These data suggest that UV-responsive production of anthocyanins is in part a consequence of the increase in carbon supply via promoting the shikimate pathway and the Phe/Trp specific pathway.