ß-sitosterol from Clinacanthus nutans Lindau enhances osteoblastogenic activity via upregulation of differentiation related genes and proteins.

ß-sitosterol derived from Clinacanthus nutans Lindau was tested for its in vitro osteogenic activity using MC3T3-E1 pre-osteoblasts. Our results indicated that ß-sitosterol was non-toxic to the cells cultured at a concentration <20 µg/mL. Treatment of the cells with ß-sitosterol significantly enhanced the alkaline phosphatase activity up to 210 and 204.6% at 5 and 10 µg/mL, respectively (P < .05). Similarly, the mineralization activity of the ß-sitosterol treated cells was elevated up to 134, 168, 118% at a concentration of 2.5, 5, and 10 µg/mL, respectively (P < .05). In addition, this compound up-regulated several marker genes for osteoblast differentiation, including runx2, osx and col I to 2, 2.5 and 5.6 folds at 10 µg/mL, respectively (P < .05). The expression of p38 and ERK proteins involved in the MAPK signal pathway related to mineralization and differentiation was also enhanced. Thus, the osteoblastogenic activity of ß-sitosterol was fully illustrated for the first time.

A new species of Leptolalax (Anura: Megophryidae) from northern Vietnam.

We describe a new, medium-sized Leptolalax species from Vietnam. Leptolalax petrops sp. nov. is distinguished from its congeners by a combination of having a medium-sized body (23.6-27.6 mm in 21 adult males, 30.3-47.0 mm in 17 adult females), immaculate white chest and belly, no distinct black markings on the head, highly tuberculate skin texture, toes lacking webbing and with narrow lateral fringes, and a call consisting of an average of four notes and a dominant frequency of 5.6-6.4 kHz (at 24.5-25.3 °C). Uncorrected sequence divergences between L. petrops sp. nov. and all homologous DNA sequences available for the 16S rRNA gene are >8%.

Five new, microendemic Asian Leaf-litter Frogs (Leptolalax) from the southern Annamite mountains, Vietnam.

The Leptolalax applebyi group of Asian leaf-litter frogs currently comprises four species of particularly small-bodied (<40 mm SVL) species distributed in the Central Highlands of Vietnam and northeastern Cambodia. In addition to their small size, the group is characterized by their morphological and genetic similarities, as well as their breeding habitat at headwaters of small mountain streams and seeps. A recent study suggested that at least two-thirds of the diversity of the group remained hidden within morphologically cryptic lineages. We expand upon the molecular, morphometric, and acoustic data and formally delineate and describe five of these lineages as distinct species: Leptolalax ardens sp. nov., Leptolalax kalonensis sp. nov., Leptolalax pallidus sp. nov., Leptolalax maculosus sp. nov., and Leptolalax tadungensis sp. nov. Due to habitat loss, the current ranges of these species are likely to be a fraction of their historical extent and under continued threat from deforestation.

Undiagnosed cryptic diversity in small, microendemic frogs (Leptolalax) from the Central Highlands of Vietnam.

A major obstacle in prioritizing species or habitats for conservation is the degree of unrecognized diversity hidden within complexes of morphologically similar, "cryptic" species. Given that amphibians are one of the most threatened groups of organisms on the planet, our inability to diagnose their true diversity is likely to have significant conservation consequences. This is particularly true in areas undergoing rapid deforestation, such as Southeast Asia. The Southeast Asian genus Leptolalax is a group of small-bodied, morphologically conserved frogs that inhabit the forest-floor. We examined a particularly small-bodied and morphologically conserved subset, the Leptolalax applebyi group, using a combination of molecular, morphometric, and acoustic data to identify previously unknown diversity within. In order to predict the geographic distribution of the group, estimate the effects of habitat loss and assess the degree of habitat protection, we used our locality data to perform ecological niche modelling using MaxEnt. Molecular (mtDNA and nuDNA), acoustic and subtle morphometric differences revealed a significant underestimation of diversity in the L. applebyi group; at least two-thirds of the diversity may be unrecognised. Patterns of diversification and microendemism in the group appear driven by limited dispersal, likely due to their small body size, with several lineages restricted to watershed basins. The L. applebyi group is predicted to have historically occurred over a large area of the Central Highlands of Vietnam, a considerable portion of which has already been deforested. Less than a quarter of the remaining forest predicted to be suitable for the group falls within current protected areas. The predicted distribution of the L. applebyi group extends into unsurveyed watershed basins, each potentially containing unsampled diversity, some of which may have already been lost due to deforestation. Current estimates of amphibian diversity based on morphology alone are misleading, and accurate alpha taxonomy is essential to accurately prioritize conservation efforts.