Acarbose improved survival for Apc+/Min mice

Acarbose blocks the digestion of complex carbohydrates, and the NIA Intervention Testing Program (ITP) found that it improved survival when fed to mice. Yet, we do not know if lifespan extension was caused by its effect on metabolism with regard to the soma or cancer suppression. Cancer caused death for ~80% of ITP mice. The ITP found rapamycin, an inhibitor to the pro‐growth mTORC1 (mechanistic target of rapamycin complex 1) pathway, improved survival and it suppressed tumors in Apc^+/Min mice providing a plausible rationale to ask if acarbose had a similar effect. Apc^+/Min is a mouse model prone to intestinal polyposis and a mimic of familial adenomatous polyposis in people. Polyp‐associated anemia contributed to their death. To address this knowledge gap, we fed two doses of acarbose to Apc^+/Min mice. Acarbose improved median survival at both doses. A cross‐sectional analysis was performed next. At both doses, ACA fed mice exhibited reduced intestinal crypt depth, weight loss despite increased food consumption and reduced postprandial blood glucose and plasma insulin, indicative of improved insulin sensitivity. Dose‐independent and dose‐dependent compensatory liver responses were observed for AMPK and mTORC1 activities, respectively. Only mice fed the high dose diet exhibited reductions in tumor number with higher hematocrits. Because low‐dose acarbose improved lifespan but failed to reduced tumors, its effects seem to be independent of cancer. These data implicate the importance of improved carbohydrate metabolism on survival.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

Purification of Monoclonal Antibodies Using High Performance Liquid Chromatography (HPLC)

Recent increases in the ability to detect low levels of immunofluorescence have shown the need for highly purified primary immunoreagents. There are now reports of purification of monoclonal antibodies using HPLC with reverse phase columns. In this study we have utilized standard size exclusion HPLC to purify both biotinylated and non-biotinylated monoclonal antibodies from hybridoma culture supernatants. Results indicated that both biotinylated and non-biotinylated monoclonal antibodies retained their antigen binding capacity after purification, and were not different in this capacity from commercially available, affinity purified reagents. These findings indicate that size exclusion HPLC may be used in the purification of biologically active monoclonal antibodies, and suggest that this technique may be used in the large scale production of antibodies and their fragments, in antibody purification from ascites fluid, and in antisera quality control.     

A Computational Model of Lipopolysaccharide-Induced Nuclear Factor Kappa B Activation: A Key Signalling Pathway in Infection-Induced Preterm Labour

Preterm birth is the single biggest cause of significant neonatal morbidity and mortality, and the incidence is rising. Development of new therapies to treat and prevent preterm labour is seriously hampered by incomplete understanding of the molecular mechanisms that initiate labour at term and preterm. Computational modelling provides a new opportunity to improve this understanding. It is a useful tool in (i) identifying gaps in knowledge and informing future research, and (ii) providing the basis for an in silico model of parturition in which novel drugs to prevent or treat preterm labour can be “tested”. Despite their merits, computational models are rarely used to study the molecular events initiating labour. Here, we present the first attempt to generate a dynamic kinetic model that has relevance to the molecular mechanisms of preterm labour. Using published data, we model an important candidate signalling pathway in infection-induced preterm labour: that of lipopolysaccharide (LPS) -induced activation of Nuclear Factor kappa B. This is the first model of this pathway to explicitly include molecular interactions upstream of Nuclear Factor kappa B activation. We produced a formalised graphical depiction of the pathway and built a kinetic model based on ordinary differential equations. The kinetic model accurately reproduced published in vitro time course plots of Lipopolysaccharide-induced Nuclear Factor kappa B activation in mouse embryo fibroblasts. In this preliminary work we have provided proof of concept that it is possible to build computational models of signalling pathways that are relevant to the regulation of labour, and suggest that models that are validated with wet-lab experiments have the potential to greatly benefit the field.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

Evidence for interplay between genes and maternal stress in utero: monoamine oxidase A polymorphism moderates effects of life events during pregnancy on infant negative emotionality at 5 weeks

The low activity variant of the monoamine oxidase A (MAOA) functional promoter polymorphism, MAOA‐LPR, in interaction with adverse environments (G × E) is associated with child and adult antisocial behaviour disorders. MAOA is expressed during foetal development so in utero G × E may influence early neurodevelopment. We tested the hypothesis that MAOA G × E during pregnancy predicts infant negative emotionality soon after birth. In an epidemiological longitudinal study starting in pregnancy, using a two stage stratified design, we ascertained MAOA‐LPR status (low vs. high activity variants) from the saliva of 209 infants (104 boys and 105 girls), and examined predictions to observed infant negative emotionality at 5 weeks post‐partum from life events during pregnancy. In analyses weighted to provide estimates for the general population, and including possible confounders for life events, there was an MAOA status by life events interaction (P = 0.017). There was also an interaction between MAOA status and neighbourhood deprivation (P = 0.028). Both interactions arose from a greater effect of increasing life events on negative emotionality in the MAOA‐LPR low activity, compared with MAOA‐LPR high activity infants. The study provides the first evidence of moderation by MAOA‐LPR of the effect of the social environment in pregnancy on negative emotionality in infancy, an early risk for the development of child and adult antisocial behaviour disorders .     

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.