Characterization of Clostridium thermocellum Isolates Grown on Cellulose and Sugarcane Bagasse

Although plant cell walls may be degraded by microbial free enzymes, many bacteria degrade cellulose via enzyme complexes called cellulosomes. The study of the structures and mechanisms of these large macromolecular complexes is an active and ongoing research topic, with the goal of developing methods to improve lignocellulosic biomass conversion using cellulosomes. The aim of the present work was to evaluate and characterize the holocellulolytic activities produced by two new isolates (ISO1 and ISO2) of the spore-forming thermophilic anaerobic bacterium Clostridium thermocellum, during growth on crystalline cellulose and sugarcane bagasse, in comparison with activities obtained from the C. thermocellum strain CthJW. The pH and temperature values for optimal growth of the isolates were pH 7 and 60 °C, respectively. The isolates produced cellulolytic, xylanolytic, and pectinolytic activities when cultured on crystalline cellulose or sugarcane bagasse, which have never been used previously as the sole carbon source for these bacteria. The profiles of secreted proteins for these isolates, ISO1 and ISO2, were quite different from those obtained for the standard strain CthJW and from each other, as shown by 2D gel electrophoresis maps, and these profiles also depend on the carbon source used. Different protein isoforms were also detected in the maps for all growth conditions and bacterial strains. MALDI-TOF mass spectrometry was used to identify the differentially expressed proteins for ISO1 and ISO2 under growth in the presence of cellulose as carbon source. Twenty-five differentially expressed spots were identified and grouped into 8 functional categories: metabolism (20 %), motor function (20 %), protein synthesis (12 %), oxidative stress (16 %), secretory pathway (12 %), cellulose hydrolysis (4 %), protein folding (4 %), and defense (12 %). Spots 200 and 197, identified as a glycosyl hydrolase family member 9 and as a chaperone GroEL, respectively, were detected for all isolates and are potentially related to cellulosome architecture.     

Molecular and iridescent feather reflectance data reveal recent genetic diversification and phenotypic differentiation in a cloud forest hummingbird

The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue‐throated populations (Sierra Madre del Sur); two groups of amethyst‐throated populations (Trans‐Mexican Volcanic Belt and Sierra Madre Oriental); and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39–0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue‐ and amethyst‐throated populations does not correspond to the 2‐MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species’ suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest‐interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic diversity or extinction.     

Criterion-Related Validity of the Distance- and Time-Based Walk/Run Field Tests for Estimating Cardiorespiratory Fitness: A Systematic Review and Meta-Analysis

Objectives

The main purpose of the present meta-analysis was to examine the criterion-related validity of the distance- and time-based walk/run tests for estimating cardiorespiratory fitness among apparently healthy children and adults.

Materials and Methods

Relevant studies were searched from seven electronic bibliographic databases up to August 2015 and through other sources. The Hunter-Schmidt’s psychometric meta-analysis approach was conducted to estimate the population criterion-related validity of the following walk/run tests: 5,000 m, 3 miles, 2 miles, 3,000 m, 1.5 miles, 1 mile, 1,000 m, ½ mile, 600 m, 600 yd, ¼ mile, 15 min, 12 min, 9 min, and 6 min.

Results

From the 123 included studies, a total of 200 correlation values were analyzed. The overall results showed that the criterion-related validity of the walk/run tests for estimating maximum oxygen uptake ranged from low to moderate (r_p = 0.42–0.79), with the 1.5 mile (r_p = 0.79, 0.73–0.85) and 12 min walk/run tests (r_p = 0.78, 0.72–0.83) having the higher criterion-related validity for distance- and time-based field tests, respectively. The present meta-analysis also showed that sex, age and maximum oxygen uptake level do not seem to affect the criterion-related validity of the walk/run tests.

Conclusions

When the evaluation of an individual’s maximum oxygen uptake attained during a laboratory test is not feasible, the 1.5 mile and 12 min walk/run tests represent useful alternatives for estimating cardiorespiratory fitness. As in the assessment with any physical fitness field test, evaluators must be aware that the performance score of the walk/run field tests is simply an estimation and not a direct measure of cardiorespiratory fitness.     

Hummingbirds as vectors of fungal spores in Moussonia deppeana (Gesneriaceae): taking advantage of a mutualism?

Hummingbirds act as vectors of Fusarium moniliforme spores on protandrous flowers of Moussonia deppeana. The resulting interactions between the pathogen and plant-pollinator interactions were investigated in a 4-yr study to determine the pathogen's impact on host flowering phenology, flower longevity, nectar production, and fruit and seed production. We also evaluated hummingbird behavior on healthy and diseased plants and its effectiveness on spore transmission. Individual plants expressed the disease from year to year, and new infected individuals were detected every year. A fraction of the flowers in a plant expressed the disease, and this varied among and within years. Diseased plants produced more inflorescences, buds, and open healthy flowers than did healthy plants. Further, diseased plants bore proportionally fewer pistillate flowers than did healthy plants when considering only healthy flowers. Neither nectar nor fruit production differed between healthy and diseased plants, but healthy plants produced more seeds. Infected flowers were retained longer than uninfected ones, producing an additional 2 mg · μL(-1) · flower(-1) of nectar sugar. Hummingbirds visited more flowers on diseased plants than they did on healthy plants, regardless of number and sexual phase. Most pollen and spores were deposited within plants. These behavioral outcomes may promote geitonogamy and limit fungal spore mixing.     

Thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV: Structure-based drug design,synthesis, and biological evaluation

A novel series of potent thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV was discovered using structure-based drug design. Synthesis, structure–activity relationship, and optimization of physicochemical properties are described. Low nanomolar potency was achieved, and selected compounds with improved thermodynamic solubility showed promising in vitro inhibition of carbonic anhydrase activity in rabbit iris ciliary body homogenate.     

ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts

Autophagy can sustain or kill tumor cells depending upon the context. The mechanism of autophagy-associated cell death has not been well elucidated and autophagy has enhanced or inhibited sensitivity of cancer cells to cytotoxic chemotherapy in different models. ARHI (DIRAS3), an imprinted tumor suppressor gene, is downregulated in 60% of ovarian cancers. In cell culture, re-expression of ARHI induces autophagy and ovarian cancer cell death within 72 h. In xenografts, re-expression of ARHI arrests cell growth and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks, dormancy is broken and xenografts grow promptly. In this study, ARHI-induced ovarian cancer cell death in culture has been found to depend upon autophagy and has been linked to G1 cell-cycle arrest, enhanced reactive oxygen species (ROS) activity, RIP1/RIP3 activation and necrosis. Re-expression of ARHI enhanced the cytotoxic effect of cisplatin in cell culture, increasing caspase-3 activation and PARP cleavage by inhibiting ERK and HER2 activity and downregulating XIAP and Bcl-2. In xenografts, treatment with cisplatin significantly slowed the outgrowth of dormant autophagic cells after reduction of ARHI, but the addition of chloroquine did not further inhibit xenograft outgrowth. Taken together, we have found that autophagy-associated cancer cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells are still vulnerable to cisplatin-based chemotherapy.Autophagy has a well-defined role in cellular physiology, removing senescent organelles and catabolizing long-lived proteins.^{1, 2} Under nutrient-poor conditions, the fatty acids and amino acids produced by hydrolysis of lipids and proteins in autophagolysosomes can provide energy to sustain starving cells. Prolonged autophagy is, however, associated with caspase-independent type II programmed cell death. Although the mechanism of autophagy-associated cell death has not been adequately characterized, programmed necrosis or necroptosis has been implicated in some studies.^{3, 4}Given the ability to sustain or kill cells, the role of autophagy in cancer is complex and dependent on the context of individual studies. During oncogenesis in genetically engineered mice, reduced hemizygous expression of genes required for autophagy (BECN1, Atg4, ATG5, Atg7) can accelerate spontaneous or chemically induced tumor formation,^{5, 6} suggesting that autophagy can serve as a tumor suppressor. Other observations with established cancers suggest that autophagy can sustain metabolically challenged neoplasms, particularly in settings with inadequate vascular access.^{7, 8} Autophagy has also been shown to protect cancer cells from the lethal effects of some cytotoxic drugs.^{9, 10}Our group has found that cancer cell proliferation,^{11, 12, 13} motility,¹⁴ autophagy and tumor dormancy^{15, 16} can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), that is downregulated in 60% of ovarian cancers by multiple mechanisms,^{17, 18} associated with shortened progression-free survival.¹⁹ Ovarian cancer cell sublines have been developed with tet-inducible expression of ARHI. In cell culture, re-expression of ARHI induces autophagy and clonogenic ovarian cancer cell death within 72 h.¹⁶ In xenografts, re-expression of ARHI arrests cell growth, inhibits angiogenesis and induces autophagy, but does not kill engrafted cancer cells. When ARHI levels are reduced after 6 weeks of induction, dormancy is broken, vascularization occurs and xenografts grow promptly. Treatment of dormant xenografts with chloroquine (CQ), a functional inhibitor of autophagy, delays tumor outgrowth, suggesting that autophagy facilitates survival of poorly vascularized, nutrient-deprived ovarian cancer cells. The relevance of this model to human disease is supported by the recent observation that small deposits of dormant ovarian cancer found on the peritoneal surface at ‘second look'' operations following initial surgery and chemotherapy exhibit autophagy and increased expression of ARHI in >80% of cases.²⁰Ovarian cancer develops in >22 000 women each year in the United States.²¹ Over the past four decades, the 5-year survival has increased from 37% to ∼50% with optimal cytoreductive surgery and combination chemotherapy using taxane- and platinum-based regimens,^{21, 22} but long-term survival and cure stand at ∼30% for all stages, due, in large part, to the persistence and recurrence of dormant, drug-resistant ovarian cancer cells. For the past two decades, standard chemotherapy for ovarian cancer has included a combination of a platinum compound and a taxane. Carboplatin and cisplatin are alkylating agents that bind covalently to DNA producing intra- and inter-strand crosslinks that, if not repaired, induce apoptosis and cell death.^{23, 24} Our previous studies suggest that ∼20% of primary ovarian cancers exhibit punctate immunohistochemical staining for LC3, a biomarker for autophagy that decorates autophagosome membranes, whereas >80% of cancers that have survived platinum-based chemotherapy exhibit punctate LC3.²⁰ Consequently, autophagy might provide one mechanism of resistance to platinum-based therapy.In this report, we have explored mechanism(s) by which ARHI induces autophagy-associated cell death and enhances cisplatin cytotoxicity. Cisplatin has been found to trigger apoptosis by inducing caspase-3 activation and PARP cleavage in ovarian cancer cells.^{25, 26} We hypothesized that autophagy-associated cell death and autophagy-enhanced sensitivity to cisplatin depend upon different mechanisms and that dormant, autophagic cancer cells might still be vulnerable to platinum-based chemotherapy.     

Phylogeography of Podocarpus matudae (Podocarpaceae): pre‐Quaternary relicts in northern Mesoamerican cloud forests

Aim Cloud forests of northern Mesoamerica represent the northern and southern limit of the contact zone between species otherwise characteristic of North or South America. Several phylogeographic studies featuring temperate conifer species have improved our understanding of species responses to environmental changes. In contrast, conifer species that presumably colonized northern Mesoamerica from South America are far less studied. A phylogeographic study of Podocarpus matudae (Podocarpaceae) was conducted to identify any major evolutionary divergences or disjunctions across its range and to determine if its current distribution is associated with pre‐Quaternary climatic and/or long‐distance dispersal events. Location Northern Mesoamerica (Mexico and Guatemala). Methods Sixteen populations (157 individuals) of P. matudae were screened for variation at two plastid DNA markers. The intra‐specific phylogenetic relationships among haplotypes were reconstructed using Bayesian inference. Population genetic analyses were undertaken to gain insight into the evolutionary history of these populations. To test whether genetic divergence among populations occurred at different time‐scales plastid DNA sequence data and fossil‐ and coalescent‐based calibrations were integrated. Results The combination of plastid markers yielded 11 haplotypes. Differentiation among populations based on DNA variation (G_ST) (0.707, SE 0.0807) indicated a clear population structure in P. matudae. Differentiation for ordered alleles (N_ST) (0.811, SE 0.0732) was higher than that for G_ST, indicating phylogeographical structure in P. matudae. Most of the total variation (81.3%, P < 0.0001) was explained by differences among populations. The estimated divergence time between the unique haplotypes from a Guatemalan population and the two most common haplotypes from the Sierra Madre Oriental in Mexico was between 10 and 20 Ma, and further haplotype divergence in the poorly resolved clade of the Sierra Madre Oriental occurred between 3 and 0.5 Ma. Main conclusions Divergence estimations support the hypothesis that extant Podocarpus matudae populations are pre‐Quaternary relicts. This finding is consistent with fossil and pollen data that support a Miocene age for temperate floristic elements in Mesoamerican cloud forests, whereas further haplotype divergence within the Sierra Madre Oriental, Chiapas and Guatemala occurred more recently, coinciding with Pleistocene cloud forest refugia.     

Development of distylous flowers and investment of biomass in male and female function in Palicourea padifolia (Rubiaceae)

Knowledge of developmental pathways for achieving differences in style and anther heights, in concert with those of ancillary features accompanied with data in regard to biomass investment to male and female function, provide an excellent opportunity for examining the developmental correlations between primary and ancillary floral traits so as to understand the evolution of heterostyly. The ontogenetic relationships between bud length and anther height and between bud length and style height, and between bud length versus bud width, anther length, and number of pollen grains per anther for long-styled (LS) and short-styled (SS) morphs of P. PADIFOLIA are described. We also described the ontogenetic biomass allocation to male and female function and to corolla with elongation of buds harvested at regular intervals. We observed an early termination of stylar growth in SS buds, whereas LS styles steadily increased in size. Morph differences for relative growth rates were significant for anther height, anther length, and pollen number but not for bud width. Bud width and anther length had a negative allometric relationship with bud elongation. The relationship between bud length and number of pollen grains per anther was positive and morph differences in pollen number were detected at later stages of development. An increase in corolla mass involved a disproportionate allocation to the female function in SS flowers and male allocation was similar for the two morphs over the course of development. Our results are consistent with theoretical and empirical data for distylous species with an approach herkogamous ancestor, and with the more general hypothesis of ontogenetic lability of heterostyly, in which morph differences in style and anther heights are achieved in various ways. Variations observed in sexual investment between floral morphs suggest differences in sex expression during flower development.     

Y-chromosomal diversity in the population of Guinea-Bissau: a multiethnic perspective

Background  

The geographic and ethnolinguistic differentiation of many African Y-chromosomal lineages provides an opportunity to evaluate human migration episodes and admixture processes, in a pan-continental context. The analysis of the paternal genetic structure of Equatorial West Africans carried out to date leaves their origins and relationships unclear, and raises questions about the existence of major demographic phenomena analogous to the large-scale Bantu expansions. To address this, we have analysed the variation of 31 binary and 11 microsatellite markers on the non-recombining portion of the Y chromosome in Guinea-Bissau samples of diverse ethnic affiliations, some not studied before.