Baeckea frutescens L. Promotes wound healing by upregulating expression of TGF-ß, IL-1 ß, VEGF and MMP-2.

Baeckea frutescens L. has been traditionally used for treating snakebites and is known to possess antifebrile and hemostatic properties. These properties are closely related to wound healing. This study aimed to evaluate the wound healing properties of B. frutescens leaves extract (BFLE) in vitro and in vivo. The in vitro study focused on proliferation, migration, and expression of TGF-ß, IL-1ß, VEGF, and MMP-2 genes and proteins. The in vivo study included excisional wound healing, histology, and tensile strength studies. The ethanolic extract of B. frutescens (BFLE) was tested for its effects on proliferation and migration using keratinocytes (HaCaT) and fibroblasts (BJ) cells. Gene and protein expression related to wound healing were analyzed using real-time PCR and Western blot assays. The wound healing properties of BFLE were evaluated in vivo using Wistar albino rats, focusing on excisional wound healing, histology, and tensile strength studies. The BFLE displayed significant proliferative and migratory effects on keratinocytes and fibroblasts cells, while upregulating the expression of TGF-ß, IL-1ß, VEGF, and MMP-2 genes and proteins. BFLE also exhibited significant wound healing effects on Wistar albino rats' excisional wounds and improved the overall tensile strength. The results suggest that BFLE has strong wound healing properties, as demonstrated by its ability to increase keratinocytes and fibroblasts proliferation and migration, upregulate genes and proteins involved in the wound healing process, and improve wound healing rates and tensile strength. The findings of this study provide important insights into the potential use of B. frutescens as a natural wound healing agent.

Unravelling proximate cues of mass flowering in the tropical forests of South-East Asia from gene expression analyses.

Elucidating the physiological mechanisms of the irregular yet concerted flowering rhythm of mass flowering tree species in the tropics requires long-term monitoring of flowering phenology, exogenous and endogenous environmental factors, as well as identifying interactions and dependencies among these factors. To investigate the proximate factors for floral initiation of mast seeding trees in the tropics, we monitored the expression dynamics of two key flowering genes, meteorological conditions and endogenous resources over two flowering events of Shorea curtisii and Shorea leprosula in the Malay Peninsula. Comparisons of expression dynamics of genes studied indicated functional conservation of FLOWERING LOCUS T (FT) and LEAFY (LFY) in Shorea. The genes were highly expressed at least 1 month before anthesis for both species. A mathematical model considering the synergistic effect of cool temperature and drought on activation of the flowering gene was successful in predicting the observed gene expression patterns. Requirement of both cool temperature and drought for floral transition suggested by the model implies that flowering phenologies of these species are sensitive to climate change. Our molecular phenology approach in the tropics sheds light on the conserved role of flowering genes in plants inhabiting different climate zones and can be widely applied to dissect the flowering processes in other plant species.

Identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection.

GATEWAY cloning technology (Invitrogen) takes advantage of bacteriophage λ site-specific recombination. The life cycle of λ alternates between the lytic and lysogenic stages. DNA can be inserted or excised from the Escherichia coli host genome by recombination between specific sites, AttB (bacterial) and AttP (phage). This process is mediated by the λ proteins int (integrase) and xis (excisionase), and a host protein IHF (integration host factor). GATEWAY cloning technology uses this process to insert fragments of DNA directionally into specially adapted vectors. These vectors contain a negative selectable marker, the ccdB gene, to select against nonrecombinant clones. Promoter or gene fragments are made GATEWAY compatible with adapter primers and amplified by PCR. These fragments are used in a BP clonase reaction to create ENTRY clones. Usually the pDONR vector used to generate such ENTRY clones is chosen so that the antibiotic selection marker is different from that of the pDEST vector, which finally generates an expression clone. This favors the selection of the expression clone and selects against the pENTRY clone. Now that many pENTRY and pDEST vectors have been generated and made available in stock centers, the antibiotic resistance genes are predetermined and may not always be compatible with each other. This problem is frequently experienced by plant researchers, since many full-length cDNA libraries have been generated using the pDONR-TOPO, pDONR221, or pENTR1A vectors, which are all kanamycin resistant in bacteria, and many pDEST vectors have been adapted from conventional plant transformation vectors, which are also frequently kanamycin resistant in bacteria. The following protocol describes ways in which such difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances.