Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.     

Parental provisioning and predation risk in rhinoceros auklets (Cerorhinca monocerata): effects on nestling growth and fledging

We examined the effects of predation risk on the behavior ofrhinoceros auklets (Cerorhinca monocerata) breeding at PineIsland, British Columbia, in 1990. Provisioning parents in someareas of the colony risked predation by bald eagles (Haliacetusleucocephalus). Chicks in high and low predation risk areasof the colony hatched on approximately the same date, receivedsimilar amounts of food to 46 days of age, grew at the samerate, reached similar peak masses, and fledged at similar masses.However, chicks in high predation areas fledged at a youngerage than did chicks in low predation areas. These data are consistentwith the hypothesis that parents in high risk areas terminatedprovisioning several days before those in lower risk areas.Mass at fledging was inversely related to age at fledging inboth high and low risk areas. The regression line for the highrisk habitats lies below that from the low risk habitats, aspredicted by a model that examines optimal time of fledgingfrom the perspective of the parents. We conclude that risk ofpredation represents a significant cost of reproduction to somerhinoceros auklets and that individual auklets within the colonyvary their behavior according to predation risk.     

Structural variation of mitochondrial genomes sheds light on evolutionary history of soybeans

The architecture and genetic diversity of mitogenome (mtDNA) are largely unknown in cultivated soybean (Glycine max), which is domesticated from the wild progenitor, Glycine soja, 5000 years ago. Here, we de novo assembled the mitogenome of the cultivar ‘Williams 82’ (Wm82_mtDNA) with Illumina PE300 deep sequencing data, and verified it with polymerase chain reaction (PCR) and Southern blot analyses. Wm82_mtDNA maps as two autonomous circular chromosomes (370 871-bp Chr-m1 and 62 661-bp Chr-m2). Its structure is extensively divergent from that of the mono-chromosomal mitogenome reported in the landrace ‘Aiganhuang’ (AGH_mtDNA). Synteny analysis showed that the structural variations (SVs) between two genomes are mainly attributed to ectopic and illegitimate recombination. Moreover, Wm82_mtDNA and AGH_mtDNA each possess six and four specific regions, which are absent in their counterparts and likely result from differential sequence-loss events. Mitogenome SV was further studied in 39 wild and 182 cultivated soybean accessions distributed world-widely with PCR/Southern analyses or a comparable in silico analysis. The results classified both wild and cultivated soybeans into five cytoplasmic groups, named as GSa–GSe and G1–G5; ‘Williams 82’ and ‘Aiganhuang’ belong to G1 and G5, respectively. Notably, except for members in GSe and G5, all accessions carry a bi-chromosomal mitogenome with a common Chr-m2. Phylogenetic analyses based on mtDNA structures and chloroplast gene sequences both inferred that G1–G3, representing >90% of cultigens, likely inherited cytoplasm from the ancestor of domestic soybean, while G4 and G5 likely inherited cytoplasm from wild soybeans carrying GSa- and GSe-like cytoplasm through interspecific hybridization, offering new insights into soybean cultivation history.     

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive ¹³C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive ¹³C-methylation is a chemical reaction performed under mild conditions that modifies a protein''s primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to ¹³C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive ¹³C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein ¹³C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of ¹³C-labeling at each primary amino group. One limitation of the method arises when the protein''s N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the ¹³C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in ¹H-¹³C heteronuclear single-quantum coherence spectra.     

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.     

Assessment of coronary flow reserve in nuclear cardiology

The coronary flow reserve is a quantitative parameter defined by the ratio maximal myocardial blood flow to rest myocardial blood flow, which allows to give functional information on the whole coronary arterial tree, integrating both epicardial arteries and microcirculatory. The coronary flow reserve is a powerful tool to guide therapy and to assess prognosis. Exploratory tools, initially limited to experimental invasive techniques, have evolved over the last 10 years, allowing to envisage its use in daily clinical practice. This article reviews the pathophysiology of the coronary flow reserve and the various invasive and non-invasive exploration tools available to practitioners, integrating them into clinical practice.     

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1) selection of fluorescent dyes having minimal spectral overlap is complicated, and 2) quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH³ resin composites. Specimens were subsequently immersed for 60 sec in either Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; α=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p<0.05) on both resin composites, whereas LTO did not produce differences (p>0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.     

P50 Sensory Gating in Infants

Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating.Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e. the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits.Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.     

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g. dichloromethane, DCM).