An Image Processing Algorithm for Facile and Reproducible Quantification of Vomocytosis.

Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell. Manual quantification of time-lapse microscopy videos is currently used as the standard to study pathogen behavior and vomocytosis incidence. However, human-driven quantification of vomocytosis (and the closely related phenomenon, exocytosis) is incredibly burdensome, especially when a large volume of cells and interactions needs to be analyzed. In this study, we designed a MATLAB algorithm that measures the extent of colocalization between the phagocyte and fungal cell (Cryptococcus neoformans; CN) and rapidly reports the occurrence of vomocytosis in a high throughput manner. Our code processes multichannel, time-lapse microscopy videos of cocultured CN and immune cells that have each been fluorescently stained with unique dyes and provides quantitative readouts of the spatiotemporally dynamic process that is vomocytosis. This study also explored metrics, such as the rate of change of pathogen colocalization with the host cell, that could potentially be used to predict vomocytosis occurrence based on the quantitative data collected. Ultimately, the algorithm quantifies vomocytosis events and reduces the time for video analysis from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously minimizing human error. This tool significantly minimizes the vomocytosis analysis pipeline, accelerates our ability to elucidate unstudied aspects of this phenomenon, and expedites our ability to characterize CN strains for the study of their epidemiology and virulence.

Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells.

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined 'vomocytosis'. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition-Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as 'vomocytes'. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.

Autonomous Internal Reflux of Magnetic Nanoparticle Chains in a Flow Channel for Efficient Detection of Waterborne Bacteria.

Herein, we developed a novel method for the efficient capture of waterborne bacteria by creating an autonomous internal reflux of the magnetic nanoparticle chains (MNCs) inside a flow channel. A glass tube containing positively charged polyethyleneimine-coated MNCs (PEI-MNCs) was placed at the center of a Halbach ring, generating a strong and uniform magnetic field inside the ring. When a bacteria-spiked solution was injected into the tube, the target bacteria bound to the PEI-MNCs via an electrostatic interaction remained in the tube, whereas the unbound bacteria left the tube. Some PEI-MNC-bacteria complexes left the glass tube at high flow rates because of the drag force, which reduced the capture efficiency of the device. The loss of the PEI-MNC-bacteria complexes at high flow rates was suppressed by placing a k0 ring behind the Halbach ring. The k0 ring was used to apply a magnetic force in the opposite direction of the solution flow and create an autonomous reflux of the PEI-MNCs inside the glass tube, reducing their loss and increasing their capture efficiency. The capture efficiency of Escherichia coli O157 was determined based on the cell count to be greater than 90% at a flow rate of 15 mL/min. E. coli O157 was detected using quantitative polymerase chain reaction, and the limits of detection were 2 and 3 cfu/mL in deionized water and river water, respectively.

Recent advances in magnetic nanoparticle-based microfluidic devices for the pretreatment of pathogenic bacteria.

Rapid and sensitive detection of pathogenic bacteria in various samples, including food and drinking water, is important to prevent bacterial diseases. Most bacterial solutions contain only a small number of bacteria in complex matrices with impurities; hence, pretreatment is necessary to separate and concentrate target bacteria before sensing. Among various pretreatment methods, iron oxide magnetic nanoparticle (MNP)-based pretreatment has drawn attention owing to the unique properties of MNP, such as high magnetic susceptibility, superparamagnetism, and biocompatibility. After target bacteria are captured by recognition molecule-functionalized MNPs, bacteria-MNP complexes can be easily separated and enriched by applying an external magnetic field. Various devices, such as optical, electrochemical, and magnetoresistance sensors, can be used to detect target bacteria, and their detection principles have been discussed in numerous review papers. Herein, we focus on recent research advances and challenges in magnetic pretreatment of pathogenic bacteria using microfluidic devices, which offer the advantages of process automation and miniaturization.

Zwitterion-Coated Colloidal Magnetic Nanoparticle Clusters for Reduced Nonspecific Adsorption of Biomolecules.

This paper demonstrates fabrication of silica-shell-coated magnetic nanoparticle clusters (SMNCs) and subsequent surface engineering of SMNCs to produce surface-modified SMNCs that have zwitterionic and primary amine ligands (SMNC-ZW/Am). SMNC-ZW/Am was passivated by zwitterionic ligands for improved colloidal stability and reduced nonspecific adsorption and by primary amine ligands for facilitated conjugation with biomolecules. Hydrodynamic (HD) size and zeta potential of SMNC-ZW/Am could be flexibly tuned by controlling the relative amounts of zwitterionic and primary amine ligands. SMNC-ZW/Am had higher colloidal stability in high salt concentration and broad pH range than did bare SMNC. Nonspecific adsorption with biomolecules onto SMNC-ZW/Am surface was significantly suppressed by the zwitterionic ligands. The facile bioconjugation capability of SWNC-ZW/Am enabled conjugation of biotin and antibody to the SWNC-ZW/Am surface. Biomolecule-conjugated SMNC-ZW/Am showed specific binding affinity to streptavidin and Salmonella bacteria, with reduced nonspecific adsorption; therefore, SWMC-ZW/Am has potential use as an antifouling nanosubstrate for separation and bioanalysis.

Lactate Exposure Promotes Immunosuppressive Phenotypes in Innate Immune Cells.

INTRODUCTION: Lactate secreted by tumors is not just a byproduct, but rather an active modulator of immune cells. There are few studies aimed at investigating the true effect of lactate, which is normally confounded by pH. Such a knowledge gap needs to be addressed. Herein, we studied the immunomodulatory effects of lactate on dendritic cells (DCs) and macrophages (MΦs). METHODS: Bone marrow-derived innate immune cells were treated with 50 mM sodium lactate (sLA) and incubated for 2 days or 5 days at 37 °C. Controls included media, lipopolysaccharide (LPS), MCT inhibitors (α-cyano-4-hydroxycinnamic acid and AR-C15585). Flow cytometric analysis of immune phenotypes were performed by incubating cells with specific marker antibodies and viability dye. Differential expression analyses were conducted on R using limma-voom and adjusted p-values were generated using the Bejamini-Hochberg Procedure. RESULTS: Lactate exposure attenuated DC maturation through the downregulation of CD80 and MHCII expression under LPS stimulation. For MΦs, lactate exposure resulted in M2 polarization as evidenced by the reduction of M1 markers (CD38 and iNOS), and the increase in expression of CD163 and Arg1. We also revealed the role of monocarboxylate transporters (MCTs) in mediating lactate effect in MΦs. MCT4 inhibition significantly boosted lactate M2 polarization, while blocking of MCT1/2 failed to reverse the immunosuppressive effect of lactate, correlating with the result of gene expression that lactate increased MCT4 expression, but downregulated the expression of MCT1/2. CONCLUSIONS: This research provides valuable insight on the influence of metabolic products on tumor immunity and will help to identify novel metabolic targets for augmenting cancer immunotherapies.

Baleen-Mimicking Virtual Filters for Rapid Detection of Pathogenic Bacteria in Water Using Magnetic Nanoparticle Chains and a Halbach Ring.

We have developed a virtual filter that quickly and efficiently captures and detects pathogenic bacteria in large amounts of water. The virtual filter comprised magnetic nanoparticle chains (MNCs) obtained by cross-linking alginate-coated magnetic nanoparticles (MNPs). When the MNC solution in a disposable plastic tube was exposed to an external magnetic field, the MNCs were aligned along the magnetic field lines, forming a filter similar to a whale's baleen filtering system. A Halbach ring that increased the magnetic field inside the ring was used as the source of an external magnetic field. The Halbach ring produced a more uniform and denser alignment of MNCs than any other ring array. To demonstrate the performance of the virtual filter comprising MNCs, Escherichia coli (E. coli) O157-spiked water was injected into the virtual filter comprising polyethyleneimine-coated MNCs, and the concentration of E. coli O157 captured by electrostatic interaction was determined using qPCR analysis. The bacterial capture efficiency using MNCs was 90% at a flow rate of 5 mL/min, and the detection limit after 10 min of preconcentration of bacteria was 10 cfu/mL, which is 100 times lower than that obtained using qPCR alone.

Asymmetric Bisvinylogous Aldolation of Aldehydes via 2-Oxonia-Cope Rearrangement Enabling Total Stereochemical Control.

Highly stereoselective 2-oxonia-Cope rearrangement reactions between newly designed bisvinylogous aldolation synthons and aldehydes, which can provide ε-hydroxy-α,ß,γ,δ-unsaturated esters with excellent enantioselectivities, as well as with unprecedented E- and Z-selectivities without regioselectivity issues, are described.

Rationally Designed Chiral Synthons Enabling Asymmetric Z- and E-Selective Vinylogous Aldol Reactions of Aldehydes.

In a conceptually different approach, highly stereoselective 2-oxonia-Cope rearrangement reactions between rationally designed nonracemic vinylogous aldolation synthons and aldehydes are described to provide δ-hydroxy-α,ß-unsaturated esters with excellent enantioselectivities and, for the first time, unprecedented Z- and E-selectivities without the regioselectivity issue.

Highly Stereoselective 2-Oxonia-Cope Rearrangement: A Platform Enabling At-Will Control of Regio-, Enantio-, and Diastereoselectivity in the Vinylogous Aldol Reactions of Aldehydes.

A distinctly different approach for the vinylogous aldolation of aldehydes is described, which exploits 2-oxonia-Cope rearrangement reactions between two readily available partners, a set of rationally designed chiral homoallylic alcohol synthons and aldehydes, under simple conditions. In these processes, chirality transfer from the former to the latter is nearly perfect, giving rise to excellent enantio- and diastereoselectivity without the regioselectivity issue associated with traditional vinylogous aldol reactions.

Enantioenriched bifunctional crotylsilanes for the asymmetric synthesis of orthogonally protected 2-methyl-1,3-diols.

Enantiomerically pure α-substituted crotylsilane reagents I and ent-I undergo asymmetric aldehyde crotylation followed by Ir(I)-catalyzed diastereoselective allylic etherification to give a variety of orthogonally protected 2-methyl-1,3-diols at the synthetically useful level of yields and stereoselectivity. The reagents are air-stable and bifunctional so that they can be used in these reactions sequentially without recourse to functional group adjustments.

Air-stable bifunctional allylation reagents for the asymmetric synthesis of differentiated syn- and anti-1,3-diols.

Turning the diols: Enantiomerically pure bifunctional reagents I and ent-I undergo asymmetric aldehyde allylation followed by Ir(I)-catalyzed enantioselective decarboxylative allylic etherification to give differentiated syn- and anti-1,3-diols with complete control of the absolute and relative stereochemistry (see scheme; PMP = para-methoxyphenyl, dbcot = dibenzo[a,e]cyclooctatetraene, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene).

Ir(I)-catalyzed enantioselective decarboxylative allylic etherification: a general method for the asymmetric synthesis of aryl allyl ethers.

Ir(I)-catalyzed enantioselective decarboxylative allylic etherification of aryl allyl carbonates provides aryl allyl ethers. Key to the generality and high stereoselection of the reaction is the use of the intramolecular decarboxylative allylation process and [Ir(dbcot)Cl](2) as an Ir(I) source. Ir(I)-catalyzed diastereoselective decarboxylative allylic etherification, combined with asymmetric aldehyde crotylation and cross metathesis, can furnish monoprotected 2-methyl-1,3-diols (starting from simple aldehydes) with high diastereoselectivities.

Concise asymmetric synthesis of orthogonally protected syn- and anti-1,3-aminoalcohols.

Novel chiral binfunctional reagents V and ent-V undergo asymmetric aldehyde allylation followed by Ir(I)-catalyzed enantioselective allylic amidation to give orthogonally protected syn- and anti-1,3-aminoalcohols with complete control of absolute and relative stereochemistry. The Mitsunobu reaction of the initial homoallylic alcohol products followed by Ir(I)-catalyzed enantioselective allylic amidation provides orthogonally protected syn- and anti-1,3-diamine derivatives in high yields and with excellent stereoselectivities.

Iridium(I)-catalyzed stereospecific decarboxylative allylic amidation of chiral branched benzyl allyl imidodicarboxylates.

Ir(I)-catalyzed decarboxylative allylic amidation of chiral branched benzyl allyl imidodicarboxylates has been shown to proceed with complete retention of enantiomeric purity and configuration. The transformation is stereospecific and appears to be quite general, accommodating a wide range of R groups.

Development of more potent 4-dimethylaminopyridine analogues.

[reaction: see text] The syntheses of bicyclic diaminopyridines 3 and 4 and tricyclic triaminopyridines 5 and 6, two novel series of nucleophilic catalysts, are described. Arguments are made for predicting the superiority of these catalysts over DMAP and even 2, the best esterification catalyst reported to date. The efficiencies of DMAP, PPY, and 2-6 in catalyzing the esterification of tertiary alcohols were compared. As predicted, 5 and 6 were about 6-fold more effective than DMAP and slightly better than 2.

Stereoselective Synthesis of trans-Olefins by the Copper-Mediated S(N)2' Reaction of Vinyl Oxazines with Grignard Reagents. Asymmetric Synthesis of D-threo-Sphingosines.

The S(N)2' reaction of 6-vinyl-5,6-dihydro-4H-[1,3]oxazines with Grignard reagents in the presence of CuCN was studied, and high trans selectivity for the formation of double bond was observed with a variety of RMgX. The S(N)2' reaction, coupled with regioselective asymmetric aminohydroxylation reaction, provided a highly efficient route for the asymmetric synthesis of D-threo-N-acetylsphingosine.

Asymmetric Synthesis of (+)-Iso-6-Cassine via Stereoselective Intramolecular Amidomercuration.

The first asymmetric synthesis of (+)-iso-6-cassine is described. Lipase-catalyzed resolution, enantioselective Overman rearrangement, and diastereoselective intramolecular amidomercuration were used for the installation of the three stereocenters in (+)-iso-6-cassine, and cross-metathesis was employed for the attachment of the side-chain.