The rockefeller university

Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is approximate to 90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans. (C) 2016 Optical Society of America

PIyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PIyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PIyC holoenzyme, mediated by its PIyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PIyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PIyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PIyCB PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PIyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.

Clustered regularly interspaced short palindromic repeat (CRISPR) loci encode an adaptive immune system of prokaryotes. Within these loci, sequences intercalated between repeats known as "spacers" specify the targets of CRISPR immunity. The majority of spacers match sequences present in phages and plasmids; however, it is not known whether there are differences in the immunity provided against these diverse invaders. We studied this issue using the Staphylococcus epidermidis CRISPR system, which harbors spacers matching both phages and plasmids. We determined that this CRISPR system provides similar levels of defense against the conjugative plasmid pG0400 and the bacteriophage CNPX. However, whereas antiplasmid immunity was very sensitive to the introduction of mismatches in the target sequence, mutations in the phage target were largely tolerated. Placing the phage and plasmid targets into a vector that can be both conjugated and transduced, we demonstrated that the route of entry of the target has no impact on the effect of the mismatches on immunity. Instead, we established that the specific sequences of each spacer/target determine the susceptibility of the S. epidermidis CRISPR system to mutations. Therefore, spacers that are more resistant to mismatches would provide long-term immunity against phages and plasmids that otherwise would escape CRISPR targeting through the accumulation of mutations in the target sequence. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements. IMPORTANCE CRISPR-Cas loci protect bacteria and archaea from both phage infection and plasmid invasion. These loci harbor short sequences of phage and plasmid origin known as "spacers" that specify the targets of CRISPR-Cas immunity. The presence of a spacer sequence matching a phage or plasmid ensures host immunity against infection by these genetic elements. In turn, phages and plasmids constantly mutate their targets to avoid recognition by the spacers of the CRISPR-Cas immune system. In this study, we demonstrated that different spacer sequences vary in their ability to tolerate target mutations that allow phages and plasmids to escape from CRISPR-Cas immunity. These results uncover an unexpected complexity in the arms race between CRISPR-Cas systems and prokaryotic infectious genetic elements.

Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2(star)02 germline allele arose in multiple HIV-1 infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb-426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01 -class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01-class bNAbs and guidelines for structure-based immunogen design.

Intracellular accumulation of hyperphosphorylated tau protein is linked to neuronal degeneration in Alzheimer's disease (AD). Mounting evidence suggests that tau phosphorylation and O-N-acetylglucosamine glycosylation (O-GlcNAcylation) are mutually exclusive post-translational modifications. O-GlcNAcylation depends on 3-5% of intracellular glucose that enters the hexosamine biosynthetic pathway. To our knowledge, the existence of an imbalance between tau phosphorylation and O-GlcNAcylation has not been reported in animal models of AD, as yet. Here, we used triple trans genic (3xTg-AD) mice at 12 months, an age at which hyperphosphorylated tau is already detected and associated with cognitive decline. In these mice, we showed that tau was hyperphosphorylated on both Ser396 and Thr205 in the hippocampus, and to a lower extent and exclusively on Thr205 in the frontal cortex. Tau O-GlcNAcylation, assessed in tau immunoprecipitates, was substantially reduced in the hippocampus of 3xTg-AD mice, with no changes in the frontal cortex or in the cerebellum. No changes in the expression of the three major enzymes involved in O-GlcNAcylation, i.e., glutamine fructose-6-phosphate amidotransferase, O-linked beta-N-acetylglucosamine transferase, and O-GlcNAc hydrolase were found in the hippocampus of 3xTg-AD mice. These data demonstrate that an imbalance between tau phosphorylation and O-GlcNAcylation exists in AD mice, and strengthens the hypothesis that O-GlcNAcylation might be targeted by disease modifying drugs in AD. (C) 2016 Elsevier Ltd. All rights reserved.

Targeted, temporally regulated neural modulation is invaluable in determining the physiological roles of specific neural populations or circuits. Here we describe a system for non-invasive, temporal activation or inhibition of neuronal activity in vivo and its use to study central nervous system control of glucose homeostasis and feeding in mice. We are able to induce neuronal activation remotely using radio waves or magnetic fields via Cre-dependent expression of a GFP-tagged ferritin fusion protein tethered to the cation-conducting transient receptor potential vanilloid 1 (TRPV1) by a camelid anti-GFP antibody (anti-GFP-TRPV1)(1). Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase-Cre mice, which express Cre in glucose-sensing neurons(2). Acute activation of glucose-sensing neurons in this region increases plasma glucose and glucagon, lowers insulin levels and stimulates feeding, while inhibition reduces blood glucose, raises insulin levels and suppresses feeding. These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behaviour. The method we employ obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humor al antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals. (C) 2016 Elsevier B.V. All rights reserved.

Circadian clocks enable organisms to anticipate and adapt to fluctuating environmental conditions. Despite substantial knowledge of central clock machineries, we have less understanding of how the central clock's behavioral outputs are regulated. Here, we identify Drosophila miR-124 as a critical regulator of diurnal activity. During normal light/dark cycles, mir-124 mutants exhibit profoundly abnormal locomotor activity profiles, including loss of anticipatory capacities at morning and evening transitions. Moreover, mir-124 mutants exhibited striking behavioral alterations in constant darkness (DD), including a temporal advance in peak activity. Nevertheless, anatomical and functional tests demonstrate a normal circadian pacemaker in mir-124 mutants, indicating this miRNA regulates clock output. Among the extensive miR-124 target network, heterozygosity for targets in the BMP pathway substantially corrected the evening activity phase shift in DD. Thus, excess BMP signaling drives specific circadian behavioral output defects in mir-124 knock-outs.

Stochastic processes that are randomly reset to an initial condition serve as a showcase to investigate non-equilibrium steady states. However, all existing results have been restricted to the special case of memoryless resetting protocols. Here, we obtain the general solution for the distribution of processes in which waiting times between reset events are drawn from an arbitrary distribution. This allows for the investigation of a broader class of much more realistic processes. As an example, our results are applied to the analysis of the efficiency of constrained random search processes.

Traits of interest to evolutionary biologists often have complex genetic architectures, the nature of which can confound traditional experimental study at single levels of analysis. In the fire ant Solenopsis invicta, the presence of a Mendelian supergene' is both necessary and sufficient to induce a shift in a fundamental property of social organization, from single-queen (monogyne) to multiple-queen (polygyne) colonies. This selfish genetic element, termed the Social b (Sb) supergene, contains >600 genes that collectively promote its fitness by inducing the characteristic polygyne syndrome, in part by causing polygyne workers to accept only queens bearing the Sb element (a behaviour termed worker Sb discrimination'). Here, we employ a newly developed behavioural assay to reveal that polygyne workers, many of which bear the Sb element, employ chemical cues on the cuticle of queens to achieve worker Sb discrimination, but we found no evidence for such pheromonally mediated worker Sb discrimination in monogyne workers, which universally lack the Sb element. This polygyne worker Sb discrimination was then verified through a green beard' effect previously described in this system. We thus have demonstrated that the Sb element is required both for production of relevant chemical cues of queens and for expression of the behaviours of workers that collectively result in worker Sb discrimination. This information fills a critical gap in the map between genotype and complex phenotype in S.invicta by restricting the search for candidate genes and molecules involved in producing this complex social trait to factors associated with the Sb element itself.