The shape of abundance distributions across temperature gradients in reef fishes

Improving predictions of ecological responses to climate change requires understanding how local abundance relates to temperature gradients, yet many factors influence local abundance in wild populations. We evaluated the shape of thermal‐abundance distributions using 98 422 abundance estimates of 702 reef fish species worldwide. We found that curved ceilings in local abundance related to sea temperatures for most species, where local abundance declined from realised thermal ‘optima’ towards warmer and cooler environments. Although generally supporting the abundant‐centre hypothesis, many species also displayed asymmetrical thermal‐abundance distributions. For many tropical species, abundances did not decline at warm distribution edges due to an unavailability of warmer environments at the equator. Habitat transitions from coral to macroalgal dominance in subtropical zones also influenced abundance distribution shapes. By quantifying the factors constraining species’ abundance, we provide an important empirical basis for improving predictions of community re‐structuring in a warmer world.     

The Three Mycobacterium tuberculosis Antigen 85 Isoforms Have Unique Substrates and Activities Determined by Non-active Site Regions

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro²¹⁶–Phe²²⁸ loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.     

Prey preferences of sympatric fin (Balaenoptera physalus) and humpback (Megaptera novaeangliae) whales revealed by stable isotope mixing models

Over‐exploitation of top predators and fish stocks has altered ecosystems towards less productive systems with fewer trophic levels. In the Celtic Sea (CS), discards and bycatch levels have prompted concern about some fisheries, while fin and humpback whales are recovering from centuries of over‐exploitation. A lack of empirical evidence on the preferred diet of some predators such as whales in the CS has hindered the implementation of effective conservation measures using an ecosystem‐based approach to fisheries management. Using a Bayesian framework (SIAR), stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope mixing models were used to assign proportionate diet solutions to fin and humpback whales (skin biopsies) and putative prey items: herring (Clupea harengus), sprat (Sprattus sprattus), and krill (Meganyctiphanes norvegica and Nyctiphanes couchii) in the CS. Krill was the single most important prey item in the diet of fin whales, but one of the least important for humpback whales (albeit based on a small sample of humpback whale samples). Age 0 sprat and herring comprised a large proportion of the diet of both species, followed by older sprat (age 1–2) and older herring (age 2–4). An ecosystem based approach to fisheries management will be required in the CS if we seek effective conservation of both fin and humpback whales, and sustainable fisheries.     

Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.     

Divergent Evolution of the Activity and Regulation of the Glutamate Decarboxylase Systems in Listeria monocytogenes EGD-e and 10403S: Roles in Virulence and Acid Tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GAD_i), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GAD_i system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GAD_i/GAD_e). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GAD_i but not GAD_e the results indicate that the GAD_i system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.     

DNA Damage Responses following Exposure to Modulated Radiation Fields

During the delivery of advanced radiotherapy treatment techniques modulated beams are utilised to increase dose conformity across the target volume. Recent investigations have highlighted differential cellular responses to modulated radiation fields particularly in areas outside the primary treatment field that cannot be accounted for by scattered dose alone. In the present study, we determined the DNA damage response within the normal human fibroblast AG0-1522B and the prostate cancer cell line DU-145 utilising the DNA damage assay. Cells plated in slide flasks were exposed to 1 Gy uniform or modulated radiation fields. Modulated fields were delivered by shielding 25%, 50% or 75% of the flask during irradiation. The average number of 53BP1 or γH2AX foci was measured in 2 mm intervals across the slide area. Following 30 minutes after modulated radiation field exposure an increase in the average number of foci out-of-field was observed when compared to non-irradiated controls. In-field, a non-uniform response was observed with a significant decrease in the average number of foci compared to uniformly irradiated cells. Following 24 hrs after exposure there is evidence for two populations of responding cells to bystander signals in-and out-of-field. There was no significant difference in DNA damage response between 25%, 50% or 75% modulated fields. The response was dependent on cellular secreted intercellular signalling as physical inhibition of intercellular communication abrogated the observed response. Elevated residual DNA damage observed within out-of-field regions decreased following addition of an inducible nitric oxide synthase inhibitor (Aminoguanidine). These data show, for the first time, differential DNA damage responses in-and out-of-field following modulated radiation field delivery. This study provides further evidence for a role of intercellular communication in mediating cellular radiobiological response to modulated radiation fields and may inform the refinement of existing radiobiological models for the optimization of advanced radiotherapy treatment plans.     

An MMP13-selective inhibitor delays primary tumor growth and the onset of tumor-associated osteolytic lesions in experimental models of breast cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.     

Redundant functions of Rac GTPases in inner ear morphogenesis

Development of the mammalian inner ear requires coordination of cell proliferation, cell fate determination and morphogenetic movements. While significant progress has been made in identifying developmental signals required for inner ear formation, less is known about how distinct signals are coordinated by their downstream mediators. Members of the Rac family of small GTPases are known regulators of cytoskeletal remodeling and numerous other cellular processes. However, the function of Rac GTPases in otic development is largely unexplored. Here, we show that Rac1 and Rac3 redundantly regulate many aspects of inner ear morphogenesis. While no morphological defects were observed in Rac3(-/-) mice, Rac1(CKO); Rac3(-/-) double mutants displayed enhanced vestibular and cochlear malformations compared to Rac1(CKO) single mutants. Moreover, in Rac1(CKO); Rac3(-/-) mutants, we observed compromised E-cadherin-mediated cell adhesion, reduced cell proliferation and increased cell death in the early developing otocyst, leading to a decreased size and malformation of the membranous labyrinth. Finally, cochlear extension was severely disrupted in Rac1(CKO); Rac3(-/-) mutants, accompanied by a loss of epithelial cohesion and formation of ectopic sensory patches underneath the cochlear duct. The compartmentalized expression of otic patterning genes within the Rac1(CKO); Rac3(-/-) mutant otocyst was largely normal, however, indicating that Rac proteins regulate inner ear morphogenesis without affecting cell fate specification. Taken together, our results reveal an essential role for Rac GTPases in coordinating cell adhesion, cell proliferation, cell death and cell movements during otic development.     

Rapid identification of chloroplast haplotypes using High Resolution Melting analysis

We have evaluated High Resolution Melting (HRM) analysis as a method for one‐step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c. 360 and 390 bp) at the chloroplast rps16 intron (c. 750 bp) we applied HRM to identify haplotypes in 21 populations of two European arctic‐alpine herb species Arenaria ciliata and Arenaria norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melting‐curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR‐RFLP, only seven of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMelt^SM. In silico mutation analysis carried out on a 360 bp wild‐type rps16I amplicon determined that the expected rate of missed single‐nucleotide polymorphisms (SNP) detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. In vitro HRM successfully discriminated between all amplicon templates differing by two or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, and these samples differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single‐reaction method for haplotype identification.