The anti-ageing effects of a natural peptide discovered by artificial intelligence.

OBJECTIVE: As skin ages, impaired extracellular matrix (ECM) protein synthesis and increased action of degradative enzymes manifest as atrophy, wrinkling and laxity. There is mounting evidence for the functional role of exogenous peptides across many areas, including in offsetting the effects of cutaneous ageing. Here, using an artificial intelligence (AI) approach, we identified peptide RTE62G (pep_RTE62G), a naturally occurring, unmodified peptide with ECM stimulatory properties. The AI-predicted anti-ageing properties of pep_RTE62G were then validated through in vitro, ex vivo and proof of concept clinical testing. METHODS: A deep learning approach was applied to unlock pep_RTE62G from a plant source, Pisum sativum (pea). Cell culture assays of human dermal fibroblasts (HDFs) and keratinocytes (HaCaTs) were subsequently used to evaluate the in vitro effect of pep_RTE62G. Distinct activities such as cell proliferation and ECM protein production properties were determined by ELISA assays. Cell migration was assessed using a wound healing assay, while ECM protein synthesis and gene expression were analysed, respectively, by immunofluorescence microscopy and PCR. Immunohistochemistry of human skin explants was employed to further investigate the induction of ECM proteins by pep_RTE62G ex vivo. Finally, the clinical effect of pep_RTE626 was evaluated in a proof of concept 28-day pilot study. RESULTS: In vitro testing confirmed that pep_RTE62G is an effective multi-functional anti-ageing ingredient. In HaCaTs, pep_RTE62G treatment significantly increases both cellular proliferation and migration. Similarly, in HDFs, pep_RTE62G consistently induced the neosynthesis of ECM protein elastin and collagen, effects that are upheld in human skin explants. Lastly, in our proof of concept clinical study, application of pep_RTE626 over 28 days demonstrated anti-wrinkle and collagen stimulatory potential. CONCLUSION: pep_RTE62G represents a natural, unmodified peptide with AI-predicted and experimentally validated anti-ageing properties. Our results affirm the utility of AI in the discovery of novel, functional topical ingredients.

OBJECTIF: À mesure que la peau vieillit, une altération de la synthèse des protéines de la matrice extracellulaire (ECM) et une action accrue des enzymes dégradantes se manifestent par une atrophie, des rides et un laxisme. Il existe de plus en plus de preuves du rôle fonctionnel des peptides exogènes dans de nombreux domaines, y compris pour compenser les effets du vieillissement cutané. Ici, en utilisant une approche d'intelligence artificielle (AI), nous avons identifié le peptide RTE62G (pep_RTE62G), un peptide naturel non modifié avec des propriétés de stimulation ECM. Les propriétés anti-âge prédites par l'IA de pep_RTE62G ont ensuite été validées par des tests cliniques in vitro, ex vivo et de validation de principe. LES MÉTHODES: Une approche d'apprentissage en profondeur a été appliquée pour déverrouiller pep_RTE62G à partir d'une source végétale, Pisum sativum (pois). Des tests de culture cellulaire de fibroblastes dermiques humains (HDF) et de kératinocytes (HaCaTs) ont ensuite été utilisés pour évaluer l'effet in vitro de pep_RTE62G. Des activités distinctes telles que la prolifération cellulaire et les propriétés de production de protéines ECM ont été déterminées par des tests ELISA. La migration cellulaire a été évaluée à l'aide d'un test de cicatrisation des plaies, tandis que la synthèse des protéines ECM et l'expression des gènes ont été analysées, respectivement, par microscopie à immunofluorescence et PCR. L'immunohistochimie des explants de peau humaine a été utilisée pour approfondir l'induction des protéines ECM par pep_RTE62G ex vivo. Enfin, l'effet clinique de pep_RTE626 a été évalué dans une étude pilote de 28 jours de validation de principe. RÉSULTATS: Les tests in vitro ont confirmé que pep_RTE62G est un ingrédient anti-âge multifonctionnel efficace. Dans HaCaTs, le traitement pep_RTE62G augmente de manière significative à la fois la prolifération et la migration cellulaire. De même, dans les HDF, pep_RTE62G a induit de manière cohérente la néosynthèse de la protéine ECM élastine et collagène, effets qui sont maintenus dans les explants de peau humaine. Enfin, dans notre étude clinique de preuve de concept, l'application de pep_RTE626 sur 28 jours a démontré un potentiel stimulant anti-rides et collagène. CONCLUSION: pep_RTE62G représente un peptide naturel, non modifié avec des propriétés anti-âge prédites par l'IA et validées expérimentalement. Nos résultats confirment l'utilité de l'IA dans la découverte de nouveaux ingrédients topiques fonctionnels.

Molecular identification, incidence and phylogenetic analysis of seven viruses infecting garlic in Ethiopia.

Little information exists on the type and incidence of viruses infecting garlic (Allium sativum L) in Ethiopia. Attempts were made to identify the viruses using molecular techniques from 95 composite leaf samples collected from 44 farmers' fields and 51 germplasm accessions. Reverse transcription (RT-) PCR using genus and/or virus specific primers was used to amplify partial genome sequences of potyviruses, allexiviruses, carlaviruses and a tospovirus followed by sequencing of PCR products. Results indicated that ~73.7% of the samples are infected with at least one virus. Onion yellow dwarf virus (OYDV, genus Potyvirus, family Potyviridae) is the most common virus detected followed by Garlic virus C (genus Allexivirus) and Shallot latent virus (SLV, genus Carlavirus). Other viruses detected at lower frequency include Garlic virus X and Garlic virus D (genus Allexivirus), Leek yellow stripe virus (genus Potyvirus) and Iris yellow spot virus (IYSV, genus Tospovirus). Mixed infection of two or more viruses was detected in 65.7% of the samples. Phylogenetic analysis suggested that the different viruses may have been introduced to Ethiopia from Europe or Asia. This is the first report of Garlic virus X, Garlic virus D, IYSV and SLV in garlic in Ethiopia. The high incidence of OYDV and IYSV which cause severe yield loss alone or in mixed infection with allexiviruses and carlaviruses is a cause of concern to growers.

Whey protein isolate polydispersity affects enzymatic hydrolysis outcomes.

The effects of heat-induced denaturation of whey protein isolate (WPI) on the enzymatic breakdown of α-La, caseinomacropeptide (CMP), ß-Lg A and ß-Lg B were observed as hydrolysis proceeded to a 5% degree of hydrolysis (DH) in both unheated and heat-treated (80 °C, 10 min) WPI dispersions (100 g L(-1)). Hydrolysis of denatured WPI favoured the generation of higher levels of free essential amino acids; lysine, phenylalanine and arginine compared to the unheated substrate. LC-MS/MS identified 23 distinct peptides which were identified in the denatured WPI hydrolysate - the majority of which were derived from ß-Lg. The mapping of the detected regions in α-La, ß-Lg, and CMP enabled specific cleavage points to be associated with certain serine endo-protease activities. The outcomes of the study emphasise how a combined approach of substrate heat pre-treatment and enzymology may be used to influence proteolysis with attendant opportunities for targeting unique peptide production and amino acid release.

Rapid microsatellite marker development for African mahogany (Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity.

Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Because of heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here, we describe the development of microsatellite markers for K. senegalensis using next-generation sequencing to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next-generation sequencing yielded 93,943 sequences with an average read length of 234 bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty-one microsatellite loci were tested with 11 showing reliable amplification and polymorphism in K. senegalensis. The 11 novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa.

Abacá bunchy top virus, a new member of the genus Babuvirus (family Nanoviridae).

Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abacá (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.

Single nucleotide polymorphisms in cytochrome P450 genes from barley.

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.

Isolation and characterization of a flavonoid 3'-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida.

We have isolated a cDNA clone that corresponds to the Ht1 locus of petunia which controls the hydroxylation of dihydrokaempferol to dihydroquercetin and of naringenin to eriodictyol by the action of flavonoid 3'-hydroxylase (F3'H). The cDNA encodes a 512 amino acid polypeptide with regions of similarity to petunia flavonoid 3',5'-hydroxylases (F3'5'H). Both F3'H and F3'5'H are cytochromes P450 and are key enzymes in the flavonoid pathway leading to the production of the coloured anthocyanins. The F3 'H transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the ovaries, sepals, peduncles, stems and anthers of the petunia plant. No or very reduced levels of transcripts are detected in ht1/ht1 lines. This is the first report of isolation of a F3 'H cDNA clone from any species.

Molecular cloning and characterization of Rosa hybrida dihydroflavonol 4-reductase gene.

A full length cDNA clone encoding rose dihydroflavonol 4-reductase (DFR) was isolated from a cDNA library derived from rose petals by screening with the cDNA of Petunia hybrida DFR. Sequence comparison of the rose DFR with reported DFR genes revealed that they are homologous to each other. The amount of DFR mRNA in rose petals was developmentally regulated and paralleled anthocyanin production in petals. Sepals, thorns, styles and stamens also contained anthocyanins and DFR mRNA. No DFR mRNA was observed in mature leaves and a small amount of the transcript was detected in young leaves. A petunia cultivar, whose colour was pale pink due to a deficiency in flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase, was transformed with a binary vector containing a rose DFR cDNA cloned behind a constitutive promoter. Petals and anthers of the resultant transgenic petunia plants were salmon pink and contained pelargonidin, an anthocyanidin rarely found in petunia.

Modification of flower colour via manipulation of P450 gene expression in transgenic plants.

Unlike animals, which synthesise cytochrome P450 enzymes mostly for the degradation of xenobiotics, plants have evolved a large number of different P450 enzymes for the synthesis of secondary metabolites. Probably the most conspicuous of these secondary metabolites are anthocyanins, which are important flower pigments. The types of anthocyanins synthesised in plants are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Cloning of flavonoid 3',5'-hydroxylase genes has enabled the manipulation of anthocyanin synthesis in transgenic plants and enabled the production of novel pigments and flower colours.

Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida.

We have isolated, via differential screening of a Petunia hybrida petal cDNA library, a cDNA clone that corresponds to the Rt locus which controls the conversion of anthocyanidin-3-glucosides to anthocyanidin-3-rutinosides by the UDP rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The cDNA encodes a 469 amino acid long polypeptide with regions of similarity to the UDP glucose: flavonoid glucosyltransferases (3GT) from barley and maize. Some sequence similarity was also observed with non-plant glycosyltransferases. Two aberrant transcripts are present in most of the rt/rt petunia lines examined. Excision of a transposon from an unstable Rt locus of one petunia line (Tr38) is associated with a change in transcript size back to wild-type. The Rt transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the style and anthers but not in leaf, stem, root, petiole, ovary or sepals. Incubation of leaves in glucose under high light conditions induces the expression of the Rt gene as well as other flavonoid pathway genes. In situ hybridization revealed that the Rt transcript predominantly accumulates in the epidermal cells of the petal, the site of anthocyanin accumulation.

Cloning and expression of flavonol synthase from Petunia hybrida.

Flavonols are important co-pigments in flower colour and are also essential for pollen tube growth. In petunia, flavonol synthesis is controlled by the Fl locus. Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase family. Dioxygenase gene fragments were amplified by PCR on cDNA made from FlFl and flfl flowers using degenerate primers designed from conserved dioxygenase sequences. A petunia petal cDNA library was screened for clones that hybridized more strongly to the Fl PCR products than the fl PCR products. A full-length cDNA clone identified by this screening exhibited FLS activity when expressed in yeast. FLS gene expression is developmentally regulated during flower development. Antisense expression of an FLS cDNA clone in petunia markedly reduced flavonol synthesis in petals. RFLP mapping showed that the FLS gene is linked to Fl, suggesting that Fl is the structural gene for FLS.

Cloning and expression of cytochrome P450 genes controlling flower colour.

Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.

Structural and functional analysis of nitrogenase genes from the broad-host-range Rhizobium strain ANU240.

The genes encoding the structural components of nitrogenase, nifH, nifD and nifK, from the fast-growing, broad-host-range Rhizobium strain ANU240 have been identified and characterized. They are duplicated and linked in an operon nifHDK in both copies. Sequence analysis of the nifH gene from each copy, together with partial sequence analysis of the nifD and nifK genes, and restriction endonuclease analysis suggested that the duplication is precise. Comparison of the Fe-protein sequence from strain ANU240 with that from other nitrogen-fixing organisms revealed that, despite its broad host range and certain physiological properties characteristic of Bradyrhizobium strains, ANU240 is more closely related to the narrow-host-range Rhizobium strains than to the broad-host-range Bradyrhizobium strains. The promoter regions of both copies of the nif genes contain the consensus sequence characteristic of nif promoters, and functional analysis of the two promoters suggested that both nif operons are transcribed in nodules.

Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase.

Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase. These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences. The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit. The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit. RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene. No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2. Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product. Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E. coli RNA polymerase.

A gene cluster in the spinach and pea chloroplast genomes encoding one CF1 and three CF0 subunits of the H+-ATP synthase complex and the ribosomal protein S2.

The regions of the spinach and pea chloroplast genomes containing the ATP synthase genes atpA, atpF and atpH have been sequenced. The encoded proteins, CF1 alpha, CF0I and CF0III, are well conserved between spinach and pea, and analogous to the alpha, b and c subunits of the Escherichia coli ATP synthase complex. The atpF gene is split by a single intron, and the exon/intron boundaries have been defined by isolating and sequencing a partial cDNA clone. Two other genes, designated atpI and rps2, located upstream from atpH, have also been sequenced. They encode a 27,000 Mr hydrophobic protein analogous to the F0a subunit of E. coli ATP synthase and a basic protein analogous to the S2 protein of the E. coli 30 S ribosomal subunit. Transcriptional analysis by electron microscopy of RNA-DNA hybrids, Northern blotting and primer extension experiments shows that these genes are transcribed and processed into a complex set of transcripts, with 5' ends mapping upstream from the rps2, atpI and atpH genes.