The rockefeller university

Current atopic dermatitis (AD) models link epidermal abnormalities in lesional skin to cytokine activation. However, there is evolving evidence of systemic immune activation and detectable abnormalities in nonlesional skin. Because some of the best single correlations with severity (Scoring of AD, or SCORAD) are detected not only in lesional but also nonlesional skin and blood, more complex biomarker models of AD are needed. We thus performed extensive biomarker measures in these compartments using univariate and multivariate approaches to correlate disease biomarkers with SCORAD and with a combined hyperplasia score [ thickness and keratin 16 (K16) mRNA] at baseline and after cyclosporine A treatment in 25 moderate to severe AD patients. Increases in serum cytokines and chemokines (IL-13, IL-22, CCL17) were found in AD versus healthy individuals and were reduced with treatment. SCORAD correlated with immune (IL-13, IL-22) and epidermal (thickness, K16) measures in lesional and, even more strongly, in nonlesional AD. Serum cytokines also had higher correlations with nonlesional markers at baseline and with treatment. Multivariate U statistics improved baseline and treatment-response SCORAD correlations. Nonlesional models showed the strongest correlations, with further improvement upon integration of serum markers. Even better correlations were obtained between biomarkers and the hyperplasia score. Larger cohorts are needed to confirm these preliminary data.

Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (lambda(max) = 532 nm) with a nonrhodopsin- like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (lambda(max) = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C-15 = N isomers of the retinal cofactor with either syn-or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M-(M-2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C-13 - C-14, C-15 - N doubleisomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C-13 = C-14 double-bond isomerization.

The necessity of identifying novel methods to combat infections caused by antibiotic resistant bacteria is increasing each year. Recent advancements in the development of peptidoglycan hydrolases (e.g. lysins) from bacterial viruses (bacteriophages) have revealed the efficiency of this class of enzymes in treating serious bacterial infections. Though promising results have been obtained regarding the lethal action of lysin on bacterial pathogens both in vitro and in vivo, an often-overlooked factor in these studies is precisely identifying their peptidoglycan cleavage site. This knowledge would be useful for following the activity of the enzyme during development, without the need for whole-organism lytic assays. However, more importantly, it would enable the selection of lysins with different cleavage activities that would act synergistically for enhanced efficacy. Here, we have developed two new methods to accurately identify the cleavage site of lysins using liquid chromatography mass spectrometry (LC-MS) on peptidoglycan-like fluorophore-quencher modified synthetic peptides, as well as determining the enzymatic action and kinetics of the enzymes on modified peptides in a Forster resonance energy transfer (FRET) assay. These methods should facilitate progress within the lysin field, accelerating the development of therapeutic lysins to combat antibiotic resistant bacterial infections.

Individuals with Parkinson's disease (PD) often suffer from comorbid depression. P11 (S100A10), a member of the S100 family of proteins, is expressed widely throughout the body and is involved in major depressive disorder and antidepressant response. Central p11 levels are reduced in postmortem tissue from depressed individuals; however, p11 has not yet been investigated in PD patients with depression or those without depression. We investigated p11 levels in postmortem PD brains and assessed whether peripheral p11 levels correlate with disease severity. Substantia nigra, putamen, and cortical p11 protein levels were assessed in postmortem brain samples from PD patients and matched controls. In a different set of postmortem brains, p11 mRNA expression was measured in dopaminergic cells from the substantia nigra. Both p11 protein and mRNA levels were decreased in PD patients. Peripheral p11 protein levels were investigated in distinct leukocyte populations from PD patients with depression and those without depression. Monocyte, natural killer (NK) cell, and cytotoxic T-cell p11 levels were positively associated with the severity of PD, and NK cell p11 levels were positively associated with depression scores. Given that inflammation plays a role in both PD and depression, it is intriguing that peripheral p11 levels are altered in immune cells in both conditions. Our data provide insight into the pathological alterations occurring centrally and peripherally in PD. Moreover, if replicated in other cohorts, p11 could be an easily accessible biomarker for monitoring the severity of PD, especially in the context of comorbid depression.

The bacterial sigma factors confer promoter specificity to the RNA polymerase (RNAP). One alternative sigma factor, sigma(N), is unique in its structure and functional mechanism, forming transcriptionally inactive promoter complexes that require activation by specialized AAA(+) ATPases. We report a 3.4- angstrom resolution X-ray crystal structure of a sigma(N) fragment in complex with its cognate promoter DNA, revealing the molecular details of promoter recognition by sigma(N). The structure allowed us to build and refine an improved sigma(N)-holoenzyme model based on previously published 3.8-angstrom resolution X-ray data. The improved sigma(N)-holoenzyme model reveals a conserved interdomain interface within sigma(N) that, when disrupted by mutations, leads to transcription activity without activator intervention (so-called bypass mutants). Thus, the structure and stability of this interdomain interface are crucial for the role of sigma(N) in blocking transcription activity and in maintaining the activator sensitivity of sigma(N).

The effect of new, easy-to-use tools for editing cellular genomes in medicine and biomedical science is difficult to overstate. The new method uses programmable DNA-cutting machinery from a bacterial immune system (CRISPR [clustered regularly interspaced short palindromic repeats]–Cas9), which can be repurposed for use in nearly any type of cell. With the use of CRISPR guides (i.e., CRISPR RNA that guides Cas9 to a specific location in the human genome), the Cas9 enzyme can be targeted to inactivate or edit specific genes in cultured cells or in laboratory animals. Perhaps one day — if technical and ethical constraints are addressed — this method will be used to treat human disease.

ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55 alpha-and B56 delta-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKII alpha :: cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling.

Obesity is reaching global epidemic proportions as a result of factors such as high-calorie diets and lack of physical exercise. Obesity is now considered to be a medical condition, which not only contributes to the risk of developing type 2 diabetes mellitus, cardiovascular disease and cancer, but also negatively affects longevity and quality of life. To combat this epidemic, anti-obesogenic approaches are required that are safe, widely available and inexpensive. Several plants and mushrooms that are consumed in traditional Chinese medicine or as nutraceuticals contain antioxidants, fibre and other phytochemicals, and have anti-obesogenic and antidiabetic effects through the modulation of diverse cellular and physiological pathways. These effects include appetite reduction, modulation of lipid absorption and metabolism, enhancement of insulin sensitivity, thermogenesis and changes in the gut microbiota. In this Review, we describe the molecular mechanisms that underlie the anti-obesogenic and antidiabetic effects of these plants and mushrooms, and propose that combining these food items with existing anti-obesogenic approaches might help to reduce obesity and its complications.

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice(1-5). Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFa superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

Recent reports show transcription preference for long genes in neuronal tissues compared with non-neuronal tissues, and a gene-length dependent change in expression in the neurodevelopmental disease Rett syndrome (RTT). Whether the gene-length dependent changes in expression seen in RTT might also be seen in neurodegenerative diseases is not yet known. However, a reasonable hypothesis is that similar effects might be seen in neurodegenerative diseases as well as in RTT since a common general feature of both illnesses involves progressive dysfunction of synapses. Here, we demonstrate a clear length-dependent gene misexpression in the most prevalent neurodegenerative disease, Alzheimer's disease. We show that the effect is associated with disease progression and can be attributed specifically to neurons. In particular, we observed gene length-dependent down regulation on the level of the whole tissue and gene length-dependent up regulation on the level of single cells. Our analysis shows that a gene-length effect on expression can be found in degenerative neurological illnesses, such as Alzheimer's disease. Additional investigation to elucidate the precise mechanism underlying gene-length dependent changes in expression is warranted.