Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume

A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita

© The Author(s). 2018Background: Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. Results: A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. Conclusions: Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.Peer reviewedFinal Published versio

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells

Role of SPI-1 Secreted Effectors in Acute Bovine Response to Salmonella enterica Serovar Typhimurium: A Systems Biology Analysis Approach

Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway

SalmoNet, an integrated network of ten Salmonella enterica strains reveals common and distinct pathways to host adaptation

Salmonella enterica is a prominent bacterial pathogen with implications on human and animal health. Salmonella serovars could be classified as gastro-intestinal or extra-intestinal. Genome-wide comparisons revealed that extra-intestinal strains are closer relatives of gastro-intestinal strains than to each other indicating a parallel evolution of this trait. Given the complexity of the differences, a systems-level comparison could reveal key mechanisms enabling extra-intestinal serovars to cause systemic infections. Accordingly, in this work, we introduce a unique resource, SalmoNet, which combines manual curation, high-throughput data and computational predictions to provide an integrated network for Salmonella at the metabolic, transcriptional regulatory and protein-protein interaction levels. SalmoNet provides the networks separately for five gastro-intestinal and five extra-intestinal strains. As a multi-layered, multi-strain database containing experimental data, SalmoNet is the first dedicated network resource for Salmonella. It comprehensively contains interactions between proteins encoded in Salmonella pathogenicity islands, as well as regulatory mechanisms of metabolic processes with the option to zoom-in and analyze the interactions at specific loci in more detail. Application of SalmoNet is not limited to strain comparisons as it also provides a Salmonella resource for biochemical network modeling, host-pathogen interaction studies, drug discovery, experimental validation of novel interactions, uncovering new pathological mechanisms from emergent properties and epidemiological studies. SalmoNet is available at http://salmonet.org

Cavity-enhanced direct frequency comb spectroscopy

Cavity-enhanced direct frequency comb spectroscopy combines broad spectral bandwidth, high spectral resolution, precise frequency calibration, and ultrahigh detection sensitivity, all in one experimental platform based on an optical frequency comb interacting with a high-finesse optical cavity. Precise control of the optical frequency comb allows highly efficient, coherent coupling of individual comb components with corresponding resonant modes of the high-finesse cavity. The long cavity lifetime dramatically enhances the effective interaction between the light field and intracavity matter, increasing the sensitivity for measurement of optical losses by a factor that is on the order of the cavity finesse. The use of low-dispersion mirrors permits almost the entire spectral bandwidth of the frequency comb to be employed for detection, covering a range of ~10% of the actual optical frequency. The light transmitted from the cavity is spectrally resolved to provide a multitude of detection channels with spectral resolutions ranging from a several gigahertz to hundreds of kilohertz. In this review we will discuss the principle of cavity-enhanced direct frequency comb spectroscopy and the various implementations of such systems. In particular, we discuss several types of UV, optical, and IR frequency comb sources and optical cavity designs that can be used for specific spectroscopic applications. We present several cavity-comb coupling methods to take advantage of the broad spectral bandwidth and narrow spectral components of a frequency comb. Finally, we present a series of experimental measurements on trace gas detections, human breath analysis, and characterization of cold molecular beams.Comment: 36 pages, 27 figure

Transcriptomic profiling of host-parasite interactions in the microsporidian <i>Trachipleistophora hominis</i>

BACKGROUND: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. METHODS: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. RESULTS: T. hominis has about 30 % more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. CONCLUSIONS: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1989-z) contains supplementary material, which is available to authorized users

Pyruvate: immunonutritional effects on neutrophil intracellular amino or alpha-keto acid profiles and reactive oxygen species production

For the first time the immunonutritional role of pyruvate on neutrophils (PMN), free α-keto and amino acid profiles, important reactive oxygen species (ROS) produced [superoxide anion (O2−), hydrogen peroxide (H2O2)] as well as released myeloperoxidase (MPO) acitivity has been investigated. Exogenous pyruvate significantly increased PMN pyruvate, α-ketoglutarate, asparagine, glutamine, aspartate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Moreover, increases in O2− formation, H2O2-generation and MPO acitivity in parallel with intracellular pyruvate changes have also been detected. Regarding the interesting findings presented here we believe, that pyruvate fulfils considerably the criteria for a potent immunonutritional molecule in the regulation of the PMN dynamic α-keto and amino acid pools. Moreover it also plays an important role in parallel modulation of the granulocyte-dependent innate immune regulation. Although further research is necessary to clarify pyruvate’s sole therapeutical role in critically ill patients’ immunonutrition, the first scientific successes seem to be very promising

Platinum drugs in the treatment of non-small-cell lung cancer

The use of chemotherapy is considered standard therapy in patients with locally advanced non-small-cell lung cancer that cannot be treated with radiotherapy and in those with metastatic non-small-cell lung cancer and good performance status. This approach is also accepted in patients with earlier stage disease, when combined with radiotherapy in those with non-resectable locally advanced disease, or in the preoperative setting. Randomised clinical studies and meta-analyses of the literature have confirmed the beneficial survival effect of platinum-based chemotherapy. Cisplatin and carboplatin have been successfully used with other drugs in a wide variety of well-established two-drug combinations while three-drug combinations are still under investigation. Cisplatin and carboplatin use is limited by toxicity and inherent resistance. These considerations have prompted research into new platinum agents, such as the trinuclear platinum agent BBR3464, the platinum complex ZD0473 and oxaliplatin. These compounds could be developed in combination with agents such as paclitaxel, gemcitabine or vinorelbine in patients with advanced and/or refractory solid tumours