Conservation of the Native Orchids Through Seedling Culture and Reintroduction—A Singapore Experience

Singapore is located near the equator, off the southern tip of the Malay Peninsula. The whole country consists mostly of lowland. It has many interesting types of natural habitats such as primary rain forest, freshwater swamp forest, mangroves, secondary forests, shrub, grasslands, and urban parks and fields. The climate is equatorial with relatively uniform temperature and high humidity. Unfortunately, many of the natural habitats and the native orchids which thrive there have disappeared due to habitat destruction. Some 226 species of native orchids have been recorded in Singapore. However, of these 178 are considered to be extinct, and only five are common. The orchid conservation programme aims to monitor existing species, explore ways to conserve their germplasm, and increase their numbers in natural, semi-natural, and urban environments through ex-situ seedling culture and subsequent re-introduction into appropriate habitats, including roadside trees, parks and natural areas. In the first phase of the programme, we have successfully propagated and carried out experiments of re-introduction on five species of native orchids, namely, Grammatophyllum speciosum, Bulbophyllum vaginatum, Bulbophyllum membranaceum, Cymbidium finlaysonianum and Cymbidium bicolor. Survival percentages 8-yr after the reintroduction events ranged from 10 to 95 for G. speciosum, the target species of the earliest re-introduction experiments. Size of the seedlings at reintroduction, host trees, and relative humidity seemed to play significant roles in the success rate of the reintroductions.     

Rab35 - A vesicular traffic-regulating small GTPase with actin modulating roles

The Rab family of GTPases are regulators of eukaryotic vesicular membrane traffic, while modulation of actin dynamics is a function conventionally associated with the Rho family of GTPases. Rab35 is a Rab protein with both plasma membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions even in typically non-adherent Jurkat T-cells. Recent evidence indicates that Rab35’s activity, particularly the ability to mediate protrusive outgrowths, is due to its direct influence on actin dynamics. This can occur via activation of the Rho family of GTPases, or through the engagement of its effector fascin, an actin bundling protein.     

Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis

Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus. However, leptin receptors (LEPRs) are expressed in other sites as well, including the ventromedial hypothalamus (VMH). The possible role of leptin acting in "nonarcuate" sites has largely been ignored. In the present study, we show that leptin depolarizes and increases the firing rate of steroidogenic factor-1 (SF1)-positive neurons in the VMH. We also show, by generating mice that lack LEPRs on SF1-positive neurons, that leptin action at this site plays an important role in reducing body weight and, of note, in resisting diet-induced obesity. These results reveal a critical role for leptin action on VMH neurons.     

Cytokinin affects circadian-clock oscillation in a phytochrome B- and Arabidopsis response regulator 4-dependent manner

In higher plants, many developmental processes, such as photomorphogenesis and flowering, are coregulated by light and the phytohormone cytokinin. Interactions between light and cytokinin pathways are presumably mediated by common signaling intermediates. However, the molecular mechanism of these interactions remains unclear. Here, we report that cytokinin specifically induces the expression of the Arabidopsis circadian oscillator genes LATE ELONGATED HYPOCOTYL ( LHY ) and CIRCADIAN CLOCK-ASSOCIATED 1 ( CCA1 ) but represses the expression of TIMING OF CAB EXPRESSION 1 in a light-dependent manner. Consistent with these observations, cytokinin causes a shifted phase of the circadian clock. Mutant studies showed that the altered clock oscillation modulated by cytokinin is dependent on phytochrome B ( PHYB ) and Arabidopsis RESPONSE REGULATOR 4 ( ARR4 ). Whereas overexpression of LHY or CCA1 renders plants slightly more sensitive to cytokinin, phyB and a lhy/cca1 double mutant are less sensitive to the hormone. These results suggest that cytokinin affects the circadian clock oscillation in a PHYB - and ARR4 -dependent manner and that cytokinin signaling is also regulated by light-signaling components, including PHYB , LHY and CCA1 . Therefore, phyB, ARR4 and the circadian oscillator may function as signaling intermediates to integrate light and cytokinin pathways.     

Genomic instability and aging-like phenotype in the absence of mammalian SIRT6

The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.     

Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.     

Studying the thermal effects of a clinically-extracted vascular tissue during cryo-freezing

Cryosurgery is known to be a suitable treatment for unresectable tumors by employing extremely low temperature to induce cryo-lesion. However, large blood vessels in the tissue may induce insufficient freezing at the targeted area. In this work, we have developed a cryo-freezing model specifically dedicated to tumors with a complex blood vessel network taken from CT-scanned images. The model was validated with in-vitro experimental data. Adopting an appropriate mesh size, the simulated results achieved an excellent agreement with the experimental data at a maximum error of 3.4%. The validated model was applied to study an optimal cryotherapy in the treatment of a human liver. The movement of 265 K isotherm and the thermal influence of blood flow were investigated. Key results indicated that large neighboring vessels could significantly influence the shape of the ice fronts, but they exerted less impact on the development of the lethal temperature boundary. The freezing duration, distance to the vessel, blood flow rate and blood vessel size were observed to be key parameters in determining the optimal cryoablation. A case study further demonstrated that by precisely controlling the cryo-freezing process, up to 44.6% of the unintended tissue freezing could be achieved. Pragmatically, this work provides surgeons with essential information on how to precisely tune key parameters to promote greater surgical success.     

Non-classical membrane trafficking processes galore

Dogmatic views of how proteins and other cellular components may traffic within and between eukaryotic cells have been challenged in the past few years. Beyond the classical secretory/exocytic pathway and its established players, other pathways of cell surface membrane transport, generally termed “unconventional secretion,” are now better understood. More insights have also been gleaned on the roles of secreted or shedding microvesicles, either exosomal or ectosomal in origin, in unconventional secretion. Recent works have also revealed key molecular components, particularly the Golgi reassembly stacking protein (GRASP), and the importance of stress‐induced autophagy, in unconventional exocytic transport. This GRASP and autophagy‐dependent (GAD) mode appears to underlie the unconventional exocytosis of many soluble and membrane cargoes. Likewise, recent findings have revealed transport processes that contrast the classically known mitochondria import, namely vesicular transport from the mitochondria to peroxisomes and lysosomes. Mitochondria‐peroxisomal targeting of mitochondria‐derived vesicles appears to involve the retromer complex, which was classically associated with endosome‐Golgi membrane traffic. The routes of intracellular membrane transport and communications between eukaryotic organelles now appear far more complex that one would have imagined 10 years ago. J. Cell. Physiol. 227: 3722–3730, 2012. © 2012 Wiley Periodicals, Inc.     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.