Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.     

First estimates of metabolic rate in Atlantic bluefin tuna larvae

Atlantic bluefin tuna is an iconic scombrid species with a high commercial and ecological value. Despite their importance, many physiological aspects, especially during the larval stages, are still unknown. Metabolic rates are one of the understudied aspects in scombrid larvae, likely due to challenges associated to larval handling before and during respirometry trials. Gaining reliable estimates of metabolic rates is essential to understand how larvae balance their high growth needs and activity and other physiological functions, which can be very useful for fisheries ecology and aquaculture. This is the first study to (a) estimate the relationship between routine metabolic rate (RMR) and larval dry weight (DW) (mass scaling exponent) at a constant temperature of 26°C, (b) measure the RMR under light and darkness and (c) test whether the interindividual differences in the RMR are related to larval nutritional status (RNA/DNA and DNA/DW). The RMR scaled nearly isometrically with body size (b = 0.99, 0.60–31.56 mg DW) in contrast to the allometric relationship observed in most fish larvae (average b = 0.87). The results show no significant differences in larval RMR under light and darkness, suggesting similar larval activity levels in both conditions. The size explained most of the variability in RMR (97%), and nutritional condition was unrelated to the interindividual differences in routine metabolism. This is the first study to report the metabolic rates of Atlantic bluefin tuna larvae and discuss the challenges of performing bioenergetic studies with early life stages of scombrids.     

Expression of the Legume-Specific Nod Factor Receptor Proteins Alters Developmental and Immune Responses in Rice

Plant Molecular Biology Reporter - Legumes form symbiosis with rhizobia, which fix nitrogen for the benefit of host plant in return for carbon resources. Development of this unique symbiosis in...     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.     

Sarcoplasmic Reticulum K (TRIC) Channel Does Not Carry Essential Countercurrent during Ca Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca²⁺ efflux is compensated for by a simultaneous SR K⁺ influx. This influx is essential because, with no countercurrent, the SR membrane potential (V_m) would quickly (<1 ms) reach the Ca²⁺ equilibrium potential and SR Ca²⁺ release would cease. The SR K⁺ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K⁺ countercurrent during release. To better define the physiological role of the SR K⁺ channel, we compared SR Ca²⁺ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K⁺ channel function. Specifically, we reduced SR K⁺ channel conduction 35 and 88% by replacing cytosolic K⁺ for Na⁺ or Cs⁺ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca²⁺ reloading, and caffeine-evoked Ca²⁺ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K⁺ (TRIC) channels is not required to support SR Ca²⁺ release (or uptake). Because K⁺ enters the SR through RyRs during release, the SR K⁺ (TRIC) channel most likely is needed to restore trans-SR K⁺ balance after RyRs close, assuring SR V_m stays near 0 mV.     

Influence of seasonal variation on the phenology and liriodenine content of Annona lutescens (Annonaceae)

Annona lutescens Saff. (Annonaceae) grows as a native tree in Chiapas, Mexico in Tropical Dry Forest habitat. Like most Annonaceae, it biosynthesizes benzylisoquinoline alkaloids, mostly liriodenine. To determine the influence of seasonal changes in the accumulation of liriodenine, the monthly variation of liriodenine content in roots, stems and leaves of mature and young trees was observed. These parts of young and mature A. lutescens trees were collected monthly over a 1 year period and the alkaloids were extracted; the liriodenine was quantified by high-resolution liquid chromatography. The phenological stages of the species were also assessed (leaf development, flowering and fruiting) using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. The analysis of both young and mature trees showed a significant increase in the liriodenine concentration occurs within roots during the dry season, which coincides with leaf fall. A significant decrease also occurred at the beginning of the rainy season (the period of leaf growth); the liriodenine content for the next rainy season did not reach the levels of the previous dry season. The climatic variation induced phenological and physiological changes in this species.     

Short-term observations of marine bacterial and viral communities: patterns,connections and resilience

Observation of short-term temporal variation in bacterial and viral communities is important for understanding patterns of aquatic microbial diversity. We collected surface seawater once daily for 38 consecutive days with seven more samples interspersed over 40 more days at one location ∼2 km from Santa Catalina Island, California. Bacterial communities were analyzed by automated ribosomal intergenic spacer analysis (ARISA) and viral communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of the conserved T4-like myoviral gene encoding the major capsid protein (g23). Common bacterial and viral taxa were consistently dominant, and relatively few displayed dramatic increases/decreases or ‘boom/bust'' patterns that might be expected from dynamic predator-prey interactions. Association network analysis showed most significant covariations (associations) occurred among bacterial taxa or among viral taxa and there were several modular (highly-interconnected) associations (P⩽0.005). Associations observed between bacteria and viruses (P⩽0.005) occurred with a median time lag of 2 days. Regression of all pairwise Bray-Curtis similarities between samples indicated a rate of bacterial community change that slows from 2.1%–0.18% per day over a week to 2 months; the rate stays around 0.4% per day for viruses. Our interpretation is that, over the scale of days, individual bacterial and viral OTUs can be dynamic and patterned; resulting in statistical associations regarded as potential ecological interactions. However, over the scale of weeks, average bacterial community variation is slower, suggesting that there is strong community-level ecological resilience, that is, a tendency to converge towards a ‘mean'' microbial community set by longer-term controlling factors.     

Host‐plant associated genetic divergence of two Diatraea spp. (Lepidoptera: Crambidae) stemborers on novel crop plants

Diatraea lineolata and Diatraea saccharalis (Lepidoptera: Crambidae) are moths with stemboring larvae that feed and develop on economically important grasses. This study investigated whether these moths have diverged from a native host plant, corn, onto introduced crop plants including sorghum, sugarcane, and rice. Diatraea larvae were collected from these four host plants throughout the year in El Salvador and were reared on artificial diet until moths or parasitoids emerged. Adult moths were subsequently identified to species. Amplified fragment length polymorphisms (AFLPs) and mitochondrial DNA cytochrome oxidase I (COI) were used to examine whether or not there was genetic divergence of D. lineolata or D. saccharalis populations on the four host plants. Percent parasitism was also determined for each moth on its host plants. D. lineolata was collected from corn in the rainy season and sorghum in the dry season. D. saccharalis was most abundant on sugarcane in the rainy season and sorghum in the dry season. The AFLP analysis found two genetically divergent populations of both D. lineolata and D. saccharalis. Both moths had high levels of parasitism on their dominant host plant in the rainy season, yet had low levels of parasitism on sorghum in the dry season. The presence of two genotypes of both Diatraea spp. on sorghum suggest that host‐associated differentiation is occurring on this novel introduced crop plant.