Granule Mobility,Fusion Frequency and Insulin Secretion Are Differentially Affected by Insulinotropic Stimuli

The pre‐exocytotic behavior of insulin granules was studied against the background of the entirety of submembrane granules in MIN6 cells, and the characteristics were compared with the macroscopic secretion pattern and the cytosolic Ca²⁺ concentration of MIN6 pseudo‐islets at 22°C, 32°C and 37°C. The mobility of granules labeled by insulin–EGFP and the fusion events were assessed by TIRF microscopy utilizing an observer‐independent algorithm. In the z‐dimension, 40 mm K⁺ or 30 mm glucose increased the granule turnover. The effect of high K⁺ was quickly reversible. The increase by glucose was more sustained and modified the efficacy of a subsequent K⁺ stimulus. The effect size of glucose increased with physiological temperature whereas that of high K⁺ did not. The mobility in the x/y‐dimension and the fusion rates were little affected by the stimuli, in contrast to secretion. Fusion and secretion, however, had the same temperature dependence. Granules that appeared and fused within one image sequence had significantly larger caging diameters than pre‐existent granules that underwent fusion. These in turn had a different mobility than residence‐matched non‐fusing granules. In conclusion, delivery to the membrane, tethering and fusion of granules are differently affected by insulinotropic stimuli. Fusion rates and secretion do not appear to be tightly coupled.      

Role of host genetics and heat‐tolerant algal symbionts in sustaining populations of the endangered coral Orbicella faveolata in the Florida Keys with ocean warming

Identifying which factors lead to coral bleaching resistance is a priority given the global decline of coral reefs with ocean warming. During the second year of back‐to‐back bleaching events in the Florida Keys in 2014 and 2015, we characterized key environmental and biological factors associated with bleaching resilience in the threatened reef‐building coral Orbicella faveolata. Ten reefs (five inshore, five offshore, 179 corals total) were sampled during bleaching (September 2015) and recovery (May 2016). Corals were genotyped with 2bRAD and profiled for algal symbiont abundance and type. O. faveolata at the inshore sites, despite higher temperatures, demonstrated significantly higher bleaching resistance and better recovery compared to offshore. The thermotolerant Durusdinium trenchii (formerly Symbiondinium trenchii) was the dominant endosymbiont type region‐wide during initial (78.0% of corals sampled) and final (77.2%) sampling; >90% of the nonbleached corals were dominated by D. trenchii. 2bRAD host genotyping found no genetic structure among reefs, but inshore sites showed a high level of clonality. While none of the measured environmental parameters were correlated with bleaching, 71% of variation in bleaching resistance and 73% of variation in the proportion of D. trenchii was attributable to differences between genets, highlighting the leading role of genetics in shaping natural bleaching patterns. Notably, D. trenchii was rarely dominant in O. faveolata from the Florida Keys in previous studies, even during bleaching. The region‐wide high abundance of D. trenchii was likely driven by repeated bleaching associated with the two warmest years on record for the Florida Keys (2014 and 2015). On inshore reefs in the Upper Florida Keys, O. faveolata was most abundant, had the highest bleaching resistance, and contained the most corals dominated by D. trenchii, illustrating a causal link between heat tolerance and ecosystem resilience with global change.     

Heritable differences in fitness-related traits among populations of the mustard hill coral,Porites astreoides

A population''s potential for rapid evolutionary adaptation can be estimated from the amount of genetic variation in fitness-related traits. Inshore populations of the mustard hill coral (Porites astreoides) have been shown to be more tolerant to thermal stress than offshore populations, but it is unclear whether this difference is due to long-term physiological acclimatization or genetic adaptation. Here, we evaluated variation in growth rate and survival among 38 families of juvenile recruits of P. astreoides spawned by colonies originating from inshore and offshore locations. Recruits were reared in a common garden for 5 weeks and then subjected to two thermal treatments (28  and 31 °C) for 2.5 weeks. The most significant effects were detected during the first 5 weeks, before thermal stress was applied: 27–30% of variance in growth and 94% of variance in recruit survival was attributable to parental effects. Genotyping of eight microsatellite loci indicated that the high early mortality of some of the recruit families was not due to higher inbreeding. Post treatment, parental effects diminished such that only 10–15% of variance in growth rate was explained, which most likely reflects the dissipation of maternal effects. However, offshore-origin recruits still grew significantly less under elevated temperature compared with inshore-origin recruits. These differences observed in naive juvenile corals suggest that population-level variation in fitness in response to different thermal environments has a genetic basis and could represent raw material for natural selection in times of climate change.     

In Vivo Function of PTEX88 in Malaria Parasite Sequestration and Virulence

Malaria pathology is linked to remodeling of red blood cells by eukaryotic Plasmodium parasites. Central to host cell refurbishment is the trafficking of parasite-encoded virulence factors through the Plasmodium translocon of exported proteins (PTEX). Much of our understanding of its function is based on experimental work with cultured Plasmodium falciparum, yet direct consequences of PTEX impairment during an infection remain poorly defined. Using the murine malaria model parasite Plasmodium berghei, it is shown here that efficient sequestration to the pulmonary, adipose, and brain tissue vasculature is dependent on the PTEX components thioredoxin 2 (TRX2) and PTEX88. While TRX2-deficient parasites remain virulent, PTEX88-deficient parasites no longer sequester in the brain, correlating with abolishment of cerebral complications in infected mice. However, an apparent trade-off for virulence attenuation was spleen enlargement, which correlates with a strongly reduced schizont-to-ring-stage transition. Strikingly, general protein export is unaffected in PTEX88-deficient mutants that mature normally in vitro. Thus, PTEX88 is pivotal for tissue sequestration in vivo, parasite virulence, and preventing exacerbation of spleen pathology, but these functions do not correlate with general protein export to the host erythrocyte. The presented data suggest that the protein export machinery of Plasmodium parasites and their underlying mechanistic features are considerably more complex than previously anticipated and indicate challenges for targeted intervention strategies.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.     

Identification and characterization of a new family of C-type lectin-like genes from planaria Girardia tigrina

A novel family of C-type lectin-like genes, denoted multidomain free lectin (MDFL), was identified in the freshwater planaria Girardia (Dugesia) tigrina. We cloned several genes that encode proteins comprising a signal peptide and a number of consecutive C-type lectin-like domains (CTLDs) interconnected by short linker stretches. Analyses of genomic organization, CTLD amino acid sequences, and the overall architecture of these proteins indicate that planarian proteins are a separate family of C-type lectin-like proteins. These genes are expressed in specifically differentiated gland cells of planaria and the corresponding proteins are excreted as components of the planarian body surface mucus.     

Fluorescent proteins from nonbioluminescent Anthozoa species.

We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.     

2b or not 2b? 2bRAD is an effective alternative to ddRAD for phylogenomics

Restriction‐site‐associated DNA sequencing (RADseq) has become an accessible way to obtain genome‐wide data in the form of single‐nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non‐model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well‐supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony‐informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments.