Partnering for Greater Success: Local Stakeholders and Research in Tropical Biology and Conservation

Local communities are important stakeholders in resource management and conservation efforts, particularly in the developing world. Although evidence is mixed in suggesting that these resident stakeholders are optimal forest stewards, it is highly unlikely that large tracts of tropical forests will be conserved without engaging local people who depend on them daily for their livelihoods. Stakeholders, who reside in biodiverse ecosystems like tropical forests, are the largest direct users and ultimate decision-makers of forest fate, can be important investors in conservation, harbor local ecological knowledge that complements Western science and frequently have long-term legitimate claims on lands where they reside. Research partnerships with local stakeholders can increase research relevance, enhance knowledge exchange and result in greater conservation success. Different phases of the research cycle present distinct opportunities for partnership, with flexibility in timing, approaches and strategies depending on researcher and local stakeholder needs and interests. Despite being the last step in the research process, dissemination of results can be the best starting point for researchers interested in experimenting with local stakeholder engagement. Still, tropical biologists might not choose to partner with local people because of lack of institutional rewards, insufficient training in stakeholder engagement, insecure research infrastructure in community settings, and time and funding limitations. Although not appropriate in all cases and despite significant challenges, some biological scientists and research institutions have successfully engaged local stakeholders in the research process, proving mutually beneficial for investigators and local people alike and resulting in important innovations in tropical biology and conservation.     

Determination of lead content in medicinal plants by pre‐concentration flow injection analysis–flame atomic absorption spectrometry

Introduction – Although medicinal plants are widely used throughout the world, few studies have been carried out concerning the levels of heavy metal contaminants present. Such metals are highly toxic to living organisms even in low concentrations owing to their cumulative effect. The present paper describes the the development of a pre‐concentration flow injection analysis‐flame atomic absorption spectrometric system to determine the lead content in medicinal plants at the ppb level. Objective – To develop a pre‐concentration flow injection analysis‐flame atomic absorption spectrometric system to determine the lead content in medicinal plants at the ppb level. Methodology – A pre‐concentration flow system was coupled to a flame atomic absorption spectrometer. The plant samples were analysed after nitroperchloric digestion. The proposed system was optimised by evaluating the following parameters: nature, concentration and volume of the eluent solution, elution flow rate, elution efficiency, pre‐concentration flow rate and pre‐concentration time. Results – The proposed system exhibited good performance with high precision and repeatability (RSD ≤ 2.36%), excellent linearity (r = 0.9999), low sample consumption (10.5 mL per determination) and an analytical throughput of 55 samples/h. Lead concentrations ranged from 3.37 ± 0.25 to 7.03 ± 0.51 μg/g in dry material. This concentration interval is greater than that previously published in the literature. Conclusion – The inclusion of a pre‐concentration column in the flow manifold improved the sensitivity of the spectrometer. Thus, it was possible to determine the analyte at the ng/mL level in sample solutions of medicinal plants. This is a very important accomplishment, especially when the cumulative effect of heavy metals in living organisms is considered. Copyright © 2009 John Wiley & Sons, Ltd.     

Differential apoptosis markers in human keloids and hypertrophic scars fibroblasts

We previously demonstrated the increased amyloid precursor protein (APP) immunoreactivity around the site of damage after traumatic brain injury (TBI). However, the function of APP after TBI has not been evaluated. In this study, we investigated the effects of direct infusion of an anti-APP antibody into the damaged brain region on cerebral function and morphological changes following TBI in rats. Three days after TBI, there were many TUNEL-positive neurons and astrocytes around the damaged region and a significantly greater number of TUNEL-positive cells in the PBS group compared with the anti-APP group found. Seven days after TBI, there were significantly a greater number of large glial fibrillary acidic protein-positive cells, long elongated projections, and microtubule-associated protein-2-positive cells around the damaged region in the anti-APP group compared with the PBS group found. Seven days after TBI, the region of brain damage was significantly smaller and the time to arrival at a platform was significantly shorter in the anti-APP group compared with the PBS group. Furthermore, after TBI in the anti-APP group, the time to arrival at the platform recovered to that observed in uninjured sham operation group rats. These data suggest that the overproduction of APP after TBI inhibits astrocyte activity and reduces neural cell survival around the damaged brain region, which speculatively may be related to the induction of Alzheimer disease-type dementia after TBI.     

Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.As in all eukaryotic organisms, cell division in plants is strictly controlled by a concerted action of several key regulators, such as cyclin-dependent kinases and cyclins (De Veylder et al., 2007). The progression of the cell cycle from one phase to another requires the targeted degradation of selected cyclin molecules mediated by two ubiquitin-mediated proteolytic pathways. The SKP1-Cullin/F-Box protein (SCF) pathway acts in the G1-to-S phase transition by degrading the D-type cyclins and other substrate proteins (Yanagawa and Kimura, 2005). The second pathway, mediated by Anaphase Promoting Complex/Cyclosome (APC/C), regulates the sequential destruction of A- and B-type cyclins in a D-box or a KEN-box-dependent manner, resulting in chromosome segregation and exit from mitosis (Genschik et al., 1998; Pfleger and Kirschner, 2000). Evidence of the role of the APC/C in plant development comes from studies of the Arabidopsis (Arabidopsis thaliana) hobbit (hbt) mutant that shows defects in root growth. The HBT gene is required for both cell division and cell differentiation in root meristems and encodes CDC27, a core subunit of APC/C (Blilou et al., 2002; Perez-Perez et al., 2008). Cebolla et al. (1999) used the root nodule system of the model legume Medicago truncatula to study the function of an APC/C activator, CCS52, which is homologous to the yeast APC/C activator CDH1. A nodule-specific homolog of CCS52, CCS52A, was found to be required to initiate endoreduplication in the dividing cells, and its down-regulation affected nodule development, resulting in lower ploidy, reduced cell size, and inefficient rhizobial invasion and nodule maturation (Vinardell et al., 2003; Kondorosi et al., 2005). T-DNA insertions in the Arabidopsis CELL DIVISION CYCLE16 (CDC16) and APC2 genes result in gametophytic lethality due to the failure to degrade mitotic cyclins (Capron et al., 2003b; Kwee and Sundaresan, 2003). Although the completed Arabidopsis genome has allowed the identification of homologs of almost all vertebrate APC/C subunits in plants (Capron et al., 2003a), the functions of most of these subunits still remains largely unexplored.Direct links between root growth and auxin signaling have been well documented. Several Arabidopsis mutants with decreased auxin sensitivity often exhibit an overall defect in both primary and lateral root development (Hellmann and Estelle, 2002; Casimiro et al., 2003; Hellmann et al., 2003; Vanneste et al., 2005). A number of these auxin-resistant mutants belong to the SCF proteolysis pathway, supporting a role for the SCF pathway in auxin signaling (Teale et al., 2006; Benjamins and Scheres, 2008). Auxin appears to control lateral root development by promoting G1-to-S transition in selected xylem pericycle cells, perhaps by targeting KRP2, a cyclin-dependent kinase inhibitor, and E2F, an S phase inhibitor to SCF-mediated proteolysis (del Pozo et al., 2002; Himanen et al., 2002). Unlike SCF, the role of APC/C in auxin-mediated plant development is not clear. The only report that has so far integrated APC/C with auxin signaling pertains to the hbt mutant, which shows an increased resistance to exogenous auxin due to accumulation of Aux/IAAs in the roots (Blilou et al., 2002).As in lateral roots, auxin is an important player in the development of nodules on the roots of leguminous plants (Beveridge et al., 2007). Studies with auxin-responsive reporter gene constructs have shown auxin''s participation in cortical cell reactivation and initiation of nodule primordia (Mathesius et al., 1998). The exogenous application of Nod factor results in a transient inhibition of auxin transport capacity in roots of Vicia sativa (Boot et al., 1999) and Trifolium repens (Mathesius et al., 1998). Consistent with this, localized application of synthetic auxin transport inhibitors on alfalfa (Medicago sativa) roots induces pseudonodules (Hirsch et al., 1989). Complementing these findings, a more recent study in M. truncatula has demonstrated that increased auxin transport, caused by silencing the flavonoid pathway, results in reduced nodule formation in response to rhizobia (Wasson et al., 2006). Finally, hypernodulating mutants like sunn and skl show defective long-distance transport of auxin, further suggesting the importance of polar auxin transport, not only in regulating nodule induction but also in controlling nodule numbers (Prayitno et al., 2006; van Noorden et al., 2006).In this report, we investigated the role of the APC/C component CDC16 in root and nodule development in M. truncatula. CDC16 was identified via microarray analysis as a gene that was significantly induced in roots of M. truncatula following inoculation by Sinorhizobium meliloti and in nodules relative to uninoculated roots (Kuppusamy, 2005), thus encouraging further functional analysis of this gene. To overcome the problem of the gametophytic lethality resulting from CDC16 knockout, as seen from analysis of an insertional mutation in Arabidopsis (Kwee and Sundaresan, 2003) we undertook an RNA interference (RNAi) approach to partially suppress the expression of CDC16 gene in Agrobacterium rhizogenes-transformed roots of M. truncatula. We report that roots transformed with the CDC16 RNAi construct (hereafter called Mtcdc16i roots) displayed a decreased sensitivity to auxin, defective primary root growth, and fewer lateral roots. Interestingly, in response to S. meliloti, the Mtcdc16i roots showed almost 4-fold increase in number of nodules, suggesting that decreased sensitivity to auxin leads to a hypernodulation phenotype. Thus, this work highlights the importance of CDC16 in root and nodule development and indicates a possible role for this gene in auxin signaling.     

Isolation and characterization of microsatellite markers from Hibiscus rosa-sinensis (Malvaceae) and cross-species amplifications

We report on the development of 10 microsatellite markers in Hibiscus rosa-sinensis (Hrs). Three markers were obtained from sequences available in GenBank and seven were isolated using a two-step ‘primer extension’ procedure, based on the microsatellite-AFLP (M-AFLP) technique. Polymorphism was explored in 21 Hrs genotypes representing the genetic variation within commercial varieties. Inter-specific amplification was assessed on 12 Hibiscus wild species. A total of 45 and 56 alleles (ranging from 1 to 10 for each locus) was amplified respectively from the 21 Hrs varieties and among the full Hibiscus spp. genotype set. Primers and conditions for polymerase chain reaction (PCR) amplification of the detected loci are reported.     

RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development

Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference-based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Neurotrauma and mesenchymal stem cells treatment:From experimental studies to clinical trials

Mesenchymal stem cell(MSC)therapy has attracted the attention of scientists and clinicians around the world.Basic and pre-clinical experimental studies have highlighted the positive effects of MSC treatment after spinal cord and peripheral nerve injury.These effects are believed to be due to their ability to differentiate into other cell lineages,modulate inflammatory and immunomodulatory responses,reduce cell apoptosis,secrete several neurotrophic factors and respond to tissue injury,among others.There are many pre-clinical studies on MSC treatment for spinal cord injury(SCI)and peripheral nerve injuries.However,the same is not true for clinical trials,particularly those concerned with nerve trauma,indicating the necessity of more well-constructed studies showing the benefits that cell therapy can provide for individuals suffering the consequences of nerve lesions.As for clinical trials for SCI treatment the results obtained so far are not as beneficial as those described in experimental studies.For these reasons basic and pre-clinical studies dealing with MSC therapy should emphasize the standardization of protocols that could be translated to the clinical set with consistent and positive outcomes.This review is based on pre-clinical studies and clinical trials available in the literature from 2010 until now.At the time of writing this article there were 43 and 36 pre-clinical and 19 and 1 clinical trials on injured spinal cord and peripheral nerves,respectively.     

Influence of eCG and reproductive management in the resynchronization of ovulation in dairy goats

Resynchronization protocols have been proposed as a way of shortening females’ unproductive time in the flock, with good results in cattle and sheep. In goats, initial studies have shown that a second progestogen device inserted before luteolysis and pregnancy diagnosis does not interfere with the corpus luteum lifespan or functionality. This study aimed to evaluate the follicular growth, ovulation pattern and pregnancy rate after insertion of a second and new progestogen device for resynchronizing, with or without equine Chorionic Gonadotrophin (eCG), submitted to natural mating (NM) or artificial insemination (AI) to propose a viable resynchronization protocol for dairy goats. A total of 38 multiparous Saanen goats underwent a short-term progesterone protocol [six days exposed to medroxyprogesterone acetate (MAP) intravaginal sponges + 200 IU eCG and 0.12 mg of cloprostenol sodium on the 5^th day + 0.025 mg of lecirelin 34 hours after sponge withdrawal] and, on day 16th after the ovulation, received a new MAP device which was retained until day 21. At this moment females were split into four groups: G_eCG+NM – 100 IU eCG with NM; G_Sal+NM – saline solution with NM; G_eCG+AI – 100 IU eCG with AI; and G_Sal+AI – saline solution with AI. Ultrasound scans were performed every 12 h from sponge withdrawal (day 21) until 108 h after sponge withdrawal (day 25) for follicular dynamics evaluation, at 240 h (day 31) for assessing the presence of active corpus luteum, and on day 60 for pregnancy diagnosis. No differences were found regarding ovulation time, synchronization and follicle size. However, G_eCG+NM presented a greater estrus manifestation rate (100%) and pregnancy rate (62.5%) when compared to G_Sal+AI. In conclusion, resynchronization protocols in dairy goats may present satisfactory results.     

Drought leads to reproductive quiescence in smooth-billed anis: Phenotypic evidence for opportunistic breeding and reproductive readiness

Previous studies have suggested that the smooth-billed ani (Crotophaga ani, Linnaeus, 1758) breeds opportunistically following unpredictable rainfall in drought areas. To obtain proof of this phenomenon, the present study described and compared reproductive morphology and cell proliferation in the gonads of free-living smooth-billed anis during a wet season (April to June 2012) and the following dry season (July to September 2012) in a semiarid area using light and electron microscopy (transmission and scanning) and the AgNOR method. The morphological findings indicated distinct levels of reproductive activity related to seasonal changes. Morphological and morphometric analyses of the gonads confirmed intense gametogenic activity during the wet season, whereas gonadal involution occurred after rainfall ceased. The sizes of the testes and ovaries were significantly reduced compared to those in the wet season. The volumetric fraction of the seminiferous tubules in the testis decreased considerably, and no preovulatory follicles were detected in the ovary in the dry season. Moreover, the AgNOR count in the gonads revealed a significant decline in cell recruitment for gametogenesis after rainfall ceased. The histological findings indicated partial gonadal activation throughout the dry season. The analysis of the seminiferous epithelium confirmed the early testicular recrudescence phase, and sporadic postovulatory follicles indicated random ovulation during this time. The excurrent ducts and the oviduct also underwent remarkable involution in the dry season. Taken together, these findings confirm opportunistic breeding by smooth-billed anis in a semiarid habitat and suggest that gonadal recrudescence has been established as a reproductive strategy to cope with unexpected precipitation events.