The Marker State Space (MSS) Method for Classifying Clinical Samples

The development of accurate clinical biomarkers has been challenging in part due to the diversity between patients and diseases. One approach to account for the diversity is to use multiple markers to classify patients, based on the concept that each individual marker contributes information from its respective subclass of patients. Here we present a new strategy for developing biomarker panels that accounts for completely distinct patient subclasses. Marker State Space (MSS) defines “marker states” based on all possible patterns of high and low values among a panel of markers. Each marker state is defined as either a case state or a control state, and a sample is classified as case or control based on the state it occupies. MSS was used to define multi-marker panels that were robust in cross validation and training-set/test-set analyses and that yielded similar classification accuracy to several other classification algorithms. A three-marker panel for discriminating pancreatic cancer patients from control subjects revealed subclasses of patients based on distinct marker states. MSS provides a straightforward approach for modeling highly divergent subclasses of patients, which may be adaptable for diverse applications.     

Bisphenol A Impairs Hepatic Glucose Sensing in C57BL/6 Male Mice

Aims/Hypothesis

Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly.

Methods

To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA’s purported “safe dose” (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1–20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above.

Results

Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002–0.029 at glucose 5–20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA.

Conclusions/Interpretation

Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.     

TLR and NKG2D Signaling Pathways Mediate CS-Induced Pulmonary Pathologies

Long-term exposure to cigarette smoke (CS) can have deleterious effects on lung epithelial cells including cell death and the initiation of inflammatory responses. CS-induced cell injury can elaborate cell surface signals and cellular byproducts that stimulate immune system surveillance. Our previous work has shown that the expression of ligands for the cytotoxic lymphocyte activating receptor NKG2D is enhanced in patients with COPD and that the induction of these ligands in a mouse model can replicate COPD pathologies. Here, we extend these findings to demonstrate a role for the NKG2D receptor in CS-induced pathophysiology and provide evidence linking nucleic acid-sensing endosomal toll-like receptor (TLR) signaling to COPD pathology through NKG2D activation. Specifically, we show that mice deficient in NKG2D exhibit attenuated pulmonary inflammation and airspace enlargement in a model of CS-induced emphysema. Additionally, we show that CS exposure induces the release of free nucleic acids in the bronchoalveolar lavage and that direct exposure of mouse lung epithelial cells to cigarette smoke extract similarly induces functional nucleic acids as assessed by TLR3, 7, and 9 reporter cell lines. We demonstrate that exposure of mouse lung epithelial cells to TLR ligands stimulates the surface expression of RAET1, a ligand for NKG2D, and that mice deficient in TLR3/7/9 receptor signaling do not exhibit CS-induced NK cell hyperresponsiveness and airspace enlargement. The findings indicate that CS-induced airway injury stimulates TLR signaling by endogenous nucleic acids leading to elevated NKG2D ligand expression. Activation of these pathways plays a major role in the altered NK cell function, pulmonary inflammation and remodeling related to long-term CS exposure.     

Single,Double and Quadruple Alanine Substitutions at Oligomeric Interfaces Identify Hydrophobicity as the Key Determinant of Human Neutrophil Alpha Defensin HNP1 Function

HNP1 is a human alpha defensin that forms dimers and multimers governed by hydrophobic residues, including Tyr¹⁶, Ile²⁰, Leu²⁵, and Phe²⁸. Previously, alanine scanning mutagenesis identified each of these residues and other hydrophobic residues as important for function. Here we report further structural and functional studies of residues shown to interact with one another across oligomeric interfaces: I20A-HNP1 and L25A-HNP1, plus the double alanine mutants I20A/L25A-HNP1 and Y16A/F28A-HNP1, and the quadruple alanine mutant Y16A/I20A/L25A/F28A-HNP1. We tested binding to HIV-1 gp120 and HNP1 by surface plasmon resonance, binding to HIV-1 gp41 and HNP1 by fluorescence polarization, inhibition of anthrax lethal factor, and antibacterial activity using the virtual colony count assay. Similar to the previously described single mutant W26A-HNP1, the quadruple mutant displayed the least activity in all functional assays, followed by the double mutant Y16A/F28A-HNP1. The effects of the L25A and I20A single mutations were milder than the double mutant I20A/L25A-HNP1. Crystallographic studies confirmed the correct folding and disulfide pairing, and depicted an array of dimeric and tetrameric structures. These results indicate that side chain hydrophobicity is the critical factor that determines activity at these positions.     

Structural characterization of β-ketoacyl ACP synthase I bound to platencin and fragment screening molecules at two substrate binding sites

The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The β-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the β-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol. The enzyme is a dimer and based on structural and sequence conservation, harbors the same active site configuration as other β-ketoacyl-ACP synthases. The platencin binding site overlaps with the fatty acyl compound supplied by ACP, while 7-hydroxyl-coumarin and (5-thiophen-2-ylisoxazol-3-yl)methanol bind at the secondary fatty acyl binding site. These high-resolution structures, ranging between 1.25 and 1.70 å resolution, provide a basis for in silico inhibitor screening and optimization, and can aid in rational drug design by revealing the high-resolution binding interfaces of molecules at the malonyl-ACP and acyl-ACP active sites.     

Soil conservation practices for insect pest management in highly disturbed agroecosystems – a review

Decline in soil health is a serious worldwide problem that decreases complexity and stability of agricultural ecosystems, commonly making them more prone to outbreaks of herbivorous insect pests. Potato (Solanum tuberosum L., Solanaceae) and onion (Allium cepa L., Amaryllidaceae) production is currently characterized by high soil disturbance and heavy reliance on synthetic inputs, including insecticides. Evidence suggests that adopting soil conservation techniques often (but not always) increases mortality and decreases reproductive output for the major insect pests of these important vegetable crops. Known mechanisms responsible for such an effect include increases in density and activity of natural enemy populations, enhanced plant defenses, and modified physical characteristics of respective agricultural habitats. However, most research efforts focused on mulches and organic soil amendments, with additional research needed on elucidating effects and their mechanisms for conservation tillage, cover crops, and arbuscular mycorrhizae.     

Lateral thinking: CaMKII uncouples kainate receptors from mossy fibre synapses

EMBO J (2013) 32: 496–510 doi:10.1038/emboj.2012.334; published online January042013Alteration of the efficacy of excitatory synaptic transmission between neurons is a critical element in the processes of learning, memory, and behaviour. Despite decades of research aimed at elucidating basic cellular mechanisms underlying synaptic plasticity, new pathways and permutations continue to be discovered. Carta et al (2013) now show that activation of the calcium/calmodulin dependent kinase II (CaMKII) induces an unusual postsynaptic form of long-term depression (LTD) at the hippocampal mossy fibre synapse by promoting lateral diffusion of kainate receptors (KARs), a family of ionotropic glutamate receptors (iGluRs) that influence pyramidal neuron excitability. This report therefore reveals a new and mechanistically unique way of fine-tuning synaptic plasticity at this central synapse in the hippocampus.Information transfer within the nervous system is regulated at the synaptic level by diverse cellular mechanisms. Synaptic efficacy is not static (i.e., it is ‘plastic''), and the capacity to adjust the strength of communication between neurons in a network has been shown to be a critical component of diverse aspects of brain function that include many forms of behavioural learning (Martin et al, 2000). The complex means by which neurons adjust their synaptic properties in response to changes in local and global activity in the central nervous system has been the subject of intensive investigation spanning multiple decades (Malenka and Bear, 2004; Feldman, 2009). Nonetheless, new mechanisms underlying plasticity of excitatory and inhibitory synaptic transmission continue to be elucidated; these can vary depending on the experimental parameters for induction of plasticity, the particular type of synapse under investigation, and even the prior history of activation at the synapse. Long-term potentiation (LTP) and LTD of excitatory synaptic transmission are two well-known phenomena in which efficacy is increased or decreased, respectively, and at many synapses in the CNS occur through concomitant alterations in the number of postsynaptic iGluRs. The movement of excitatory receptors in and out of synapses, and more generally to and from the neuronal plasma membrane, is dictated by their association with a wide variety of scaffolding and chaperone proteins, whose interactions are often controlled by various protein kinases (Anggono and Huganir, 2012).It is generally appreciated now that long-term synaptic plasticity can be elicited by a variety of mechanisms even within a single type of synaptic connection. In addition to postsynaptic alterations in receptor content, for example, synaptic efficacy can also be tuned by regulated alterations in the probability of vesicular release of the neurotransmitter. Until recently, this presynaptic form of plasticity was thought to be the exclusive mechanism for altering excitatory synaptic strength at a morphologically unusual synapse in the hippocampus formed between large bouton-like presynaptic terminals arising from granule cell axons, or mossy fibres, and proximal dendrites on CA3 pyramidal neurons (Nicoll and Schmitz, 2005). These synaptic connections allow for single dentate granule cells to profoundly influence the likelihood of action potential firing in CA3 pyramidal neurons in a frequency-dependent manner, and for that reason have been referred to as ‘conditional detonator'' synapses (Henze et al, 2002). The precise mechanisms that lead to increased vesicular release probability following LTP-inducing stimulation of mossy fibre axons, including a potential role for retrograde signalling, remain the subject of debate, although there is general consensus that activation of presynaptic protein kinase A (PKA) is a key step in this form of synaptic plasticity (Figure 1A). Enhancing release probability impacts signalling through all three types of iGluRs present at mossy fibre synapses—AMPA, NMDA, and KARs. Recently, however, novel postsynaptic forms of mossy fibre plasticity were discovered in which induction protocols specifically increased the number of NMDA receptors (Kwon and Castillo, 2008; Rebola et al, 2008) or decreased the number of KARs (Selak et al, 2009), expanding the mechanistic repertoire at this historical site of focus of research on presynaptic LTP. Alterations in the synaptic content of particular iGluRs could serve as an additional means to fine-tune synaptic integration at the mossy fibre—CA3 synapse and therefore have important consequences for hippocampal network excitability.Open in a separate windowFigure 1Kainate receptor-dependent plasticity mechanisms at the hippocampal mossy fibre–CA3 synapse. (A) Activation of presynaptic receptors enhances glutamate release from the mossy fibre terminals. (B) A spike-timing-dependent plasticity protocol known to activate postsynaptic CaMKII results in long-term synaptic depression. CaMKII phosphorylates the GluK5 kainate receptor subunit, which uncouples the receptor from PSD-95 in the postsynaptic density. This leads to an increase in receptor mobility and diffusion away from the synapse. (C) Low-frequency stimulation of mossy fibres and activation of postsynaptic group 1 mGluRs leads to activation of PKC, which promotes the association of SNAP-25 to the GluK5 kainate receptor subunit and the subsequent endocytosis of synaptic receptors.In this issue, Carta et al (2013) identify a new postsynaptic mechanism for shaping mossy fibre plasticity that is specific to synaptic KARs, which serve to influence temporal integration of synaptic input as well as pyramidal neuron excitability through modulation of intrinsic ion channels. The authors paired postsynaptic depolarization of CA3 pyramidal neurons with a precisely timed presynaptic release of glutamate in a pattern that is known to produce LTP at many central synapses (Feldman, 2012). At mossy fibre synapses, however, this form of spike-timing-dependent plasticity (STDP) instead caused LTD of KAR-mediated excitatory synaptic potentials (KAR-LTD) while leaving AMPA receptor function unaltered (Figure 1B) (Carta et al, 2013). Using a series of genetic and pharmacological manipulations, Carta et al (2013) found that KAR-LTD was dependent upon the activation of postsynaptic KARs themselves, a rise in postsynaptic Ca²⁺, and CaMKII phosphorylation of a specific protein component of synaptic KARs, the GluK5 subunit. Unlike other mechanisms of postsynaptic mossy fibre plasticity, KAR-LTD was independent of NMDA or metabotropic glutamate receptor activation. Most surprisingly, KAR-LTD did not require receptor endocytosis from the plasma membrane, as is the case with most other forms of postsynaptic depression of excitatory transmission, including a distinct form of KAR-LTD reported previously (Selak et al, 2009) (Figure 1C). Instead, CaMKII-mediated phosphorylation of GluK5 subunits likely uncoupled receptors from the postsynaptic scaffolding protein PSD-95, which then led to enhanced lateral diffusion of KARs out of mossy fibre synapses. As KAR endocytosis was not altered in mossy fibre STDP, the activity-dependent reduction in KAR signalling was effectively limited to those receptors in the synapse. A molecular replacement strategy was employed using biolistic-based expression of mutant KARs in cultured hippocampal slices prepared from KAR knockout mice, which allowed Carta et al (2013) to corroborate their detailed biochemical studies by showing that reconstituted KAR currents in CA3 neurons expressing recombinant GluK5 phosphorylation site substitutions were unable to express KAR-LTD. In summary, KAR-mediated activation of CaMKII leads to phosphorylation of the GluK5 subunit and subsequent KAR-LTD through enhanced lateral mobility of synaptic receptors (Figure 1B).These findings are intriguing for several reasons. Most notably, they stand in stark contrast to studies in which CaMKII activation primarily triggers potentiation, rather than depression, of excitatory synaptic transmission at other synapses (Lisman et al, 2012). CaMKII recently was shown to cause diffusional trapping of AMPA receptor complexes within the postsynaptic density following phosphorylation of a closely associated auxiliary subunit, stargazin (Opazo et al, 2010), which is precisely the opposite of the effects of activation of the enzyme on KAR mobility at mossy fibre synapses. Further, these divergent consequences are both dependent upon carboxy-terminal PDZ interactions with scaffolding proteins, although in each case further research is needed to dissect out the relevant binding partners that control lateral mobility. It is of interest that KAR-LTD required synaptic activation of KARs to initiate signalling via CaMKII, which implies a tight coupling exists between KARs and the holoenzyme in the mossy fibre postsynaptic density. This observation also raises the possibility that activated CaMKII could phosphorylate other targets to effect other, yet-to-be-discovered, changes in synaptic function. Finally, the report by Carta et al expands our understanding of how excitatory synaptic transmission is fine-tuned at an important central synapse and underscores the fact that even well-trod ground (or synapses) continue to yield surprises that inform our understanding of the remarkable mechanistic diversity underlying synaptic plasticity in the CNS.     

Melatonin Entrains Free‐running Blind People According to a Physiological Dose‐response Curve

The specific circadian role proposed for endogenous melatonin production was based on a study of sighted people who took low pharmacological doses (500 µg) of this chemical signal for the “biological night”: the magnitude and direction of the induced phase shifts were dependent on what time of day exogenous melatonin was administered and were described by a phase‐response curve that turned out to be the opposite of that for light. We now report that lower (physiological) doses of up to 300 µg can entrain (synchronize) free‐running circadian rhythms of 10 totally blind subjects that would otherwise drift later each day. The resulting log‐linear dose‐response curve in the physiological range adds support for a circadian function of endogenous melatonin in humans. Efficacy of exogenous doses in the physiological range are of clinical significance for totally blind people who will need to take melatonin daily over their entire lifetimes in order to remain entrained to the 24 h day. Left untreated, their free‐running endocrine, metabolic, behavioral, and sleep/wake cycles can be almost as burdensome as not having vision.