Neutrophils and secondary infections in COVID-19 induced acute respiratory distress syndrome.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the current global pandemic and has affected more than 188 countries worldwide. Infection by the virus can have diverse clinical manifestations, with one of the most severe clinical manifestation being respiratory failure and the development of acute respiratory distress syndrome. Clinical manifestations of acute respiratory distress syndrome secondary to SARS-CoV-2 are also diverse with a lack of diagnostic tools to distinguish between primary viral infection and secondary bacterial infections. This was a single-centre, retrospective case-control study of bronchoalveolar lavage fluid cell counts, flow cytometry and culture results from mechanically ventilated patients with SARS-CoV-2 (COVID-19) pneumonia and acute respiratory distress syndrome. Neutrophils were the predominant cell type in bronchoalveolar fluid samples up to 2 weeks into mechanical ventilation. There also was a strong correlation between positive respiratory cultures and significant elevation in bronchoalveolar fluid neutrophil counts/percentages and serum C-reactive protein levels. Absolute levels of T cell subtypes correlated with reduced lung compliance measurements. Patients with SARS-CoV-2 and severe respiratory disease are at risk for secondary infections. In some COVID-19 patients, serum C-reactive protein and bronchoalveolar fluid neutrophils may be correlated with a secondary infection.

Critical temperature in feedback-controlled electromigration of gold nanostructures.

This paper presents several experiments demonstrating the need for a more nuanced picture of electromigration (EM) than that of a fixed critical junction temperature at which EM onset occurs. Our data suggests that even for a fixed cross-sectional geometry the critical junction temperature for EM, T c , varies with environmental temperature, thermal resistance of adjacent regions, and even the direction of the current flow in asymmetric structures. We have performed feedback-controlled EM on nanowires at environmental temperatures between 75 and 260 K and fit the EM onset points with a constant junction power model. We find that average fit critical power is monotonically increasing with decreasing temperature, but is decidedly nonlinear at lower temperatures. We extract and compare the corresponding T c values using several different thermal models which utilize measured values of nanowire thermal conductivity for our devices: these models all agree on a moderately increasing T c with decreasing environmental temperature. This is tentatively explained by enhanced current-driven annealing on the voltage ramp prior to EM onset which decreases structural scattering, thereby increasing the critical temperature at which wind-force-driven hopping events will achieve a critical atomic flux. We also obtain fit critical power for a series of bowtie structures of identical constriction but varying adjacent thermal resistance (R th ), and estimate that T c in the constriction varies with R th for higher resistance structures. Critical power measurements on a second series of asymmetric bowties further suggests that T c also depends on the alignment of the electron flow with the temperature gradient at the constriction.

Transport spectroscopy of single Pt impurities in silicon using Schottky barrier MOSFETs.

We investigate low temperature electron transport in silicon Schottky barrier metal-oxide-semiconductor field-effect transistors (MOSFETs), which consist of PtSi metallic source/drain electrodes. Measurements are made on approximately 23 inversion layers and resonances attributed to single impurities close to the metal/semiconductor interface are observed. We ascribe these impurities to Pt atoms that have diffused into the semiconductor channel from the contacts.

Observation of the linear stark effect in a single acceptor in Si.

The Stark splitting of a single fourfold degenerate impurity located within the built-in potential of a metal-semiconductor contact is investigated using low temperature transport measurements. A model is developed and used to analyze transport as a function of temperature, bias voltage, and magnetic field. Our data is consistent with a boron impurity. We report g factors of g_{1/2}=1.14 and g_{3/2}=1.72 and a linear Stark splitting 2Delta of 0.1 meV.

Mechanism of electron conduction in self-assembled alkanethiol monolayer devices.

Electron tunneling through self-assembled monolayers (SAMs) of alkanethiols was investigated using nanometer scale devices that allow temperature-dependent current-voltage, I(V, T), measurements. The I(V, T) measurement results show, for the first time, temperature-independent electron transport characteristics, proving direct tunneling as the transport mechanism in alkanethiol SAMs. The measured tunneling currents can be fitted with theoretical calculations using the modified rectangular barrier model of direct tunneling with a barrier height Phi(B) = 1.42 +/- 0.04 eV and a non-ideal barrier factor alpha = 0.65 +/- 0.01 (that may correspond to effective mass of 0.42 m). From the length-dependent conduction measurement on different alkanethiols of various lengths, the tunneling current exhibits exponential dependence on the molecular length, d, as I proportional, variant exp(-betad), where beta is a decay coefficient that was found to be bias-dependent and agrees with the existing theory of direct tunneling. A zero field decay coefficient beta(0) of 0.79 +/- 0.01 A(-1) was obtained.

Electrical characterization of metal-molecule-silicon junctions.

Direct assembly of molecules onto silicon surfaces is of particular interest for potential employment in hybrid organic-semiconductor devices. In the study we report here, aryl diazonium salts were used to assemble covalently bound molecular groups on a hydride-passivated, oxide-free n-type Si(111) surface. The reaction of 4-(trimethylsilylethynyl)benzenediazonium tetrafluoroborate generates a molecular layer of 4-(trimethylsilylethynyl)phenylene (TMS-EP) on the n++-Si(111) surface. The monolayer modifies the electrical properties of the interface and exhibits nonlinear current-voltage characteristics, as compared with the ohmic behavior observed from metal-n++-Si(111) junctions. The result of current-voltage measurements at variable temperatures (from 300 to 10 K) on samples made with the TMS-EP molecule does not show significant thermally-activated transport, indicating that tunneling is the dominant transport mechanism. The measured data is compared to a tunneling model.

Molecular wires, switches, and memories.

Design and measurements of molecular wires, switches, and memories offer an increased device capability with reduced elements. We report: Measurements on through-bond electronic transport properties of nanoscale metal-1,4-phenylene diisocyanide-metal junctions are reported, where nonohmic thermionic emission is the dominant process, with isocyanide-Pd showing the lowest thermionic barrier of 0.22 eV; robust and large reversible switching behavior in an electronic device that utilizes molecules containing redox centers as the active component, exhibiting negative differential resistance (NDR) and large on-off peak-to-valley ratio (PVR) are realized; erasable storage of higher conductivity states in these redox-center-containing molecular devices are observed; and a two-terminal electronically programmable and erasable molecular memory cell with long bit retention time is demonstrated.

Synthesis and preliminary testing of molecular wires and devices.

Presented here are several convergent synthetic routes to conjugated oligo(phenylene ethynylene)s. Some of these oligomers are free of functional groups, while others possess donor groups, acceptor groups, porphyrin interiors, and other heterocyclic interiors for various potential transmission and digital device applications. The syntheses of oligo(phenylene ethynylene)s with a variety of end groups for attachment to numerous metal probes and surfaces are presented. Some of the functionalized molecular systems showed linear, wire-like, current versus voltage (I(V)) responses, while others exhibited nonlinear I(V) curves for negative differential resistance (NDR) and molecular random access memory effects. Finally, the syntheses of functionalized oligomers are described that can form self-assembled monolayers on metallic electrodes that reduce the Schottky barriers. Information from the Schottky barrier studies can provide useful insight into molecular alligator clip optimizations for molecular electronics.

Analysis of yeast protein kinases using protein chips.

We have developed a novel protein chip technology that allows the high-throughput analysis of biochemical activities, and used this approach to analyse nearly all of the protein kinases from Saccharomyces cerevisiae. Protein chips are disposable arrays of microwells in silicone elastomer sheets placed on top of microscope slides. The high density and small size of the wells allows for high-throughput batch processing and simultaneous analysis of many individual samples. Only small amounts of protein are required. Of 122 known and predicted yeast protein kinases, 119 were overexpressed and analysed using 17 different substrates and protein chips. We found many novel activities and that a large number of protein kinases are capable of phosphorylating tyrosine. The tyrosine phosphorylating enzymes often share common amino acid residues that lie near the catalytic region. Thus, our study identified a number of novel features of protein kinases and demonstrates that protein chip technology is useful for high-throughput screening of protein biochemical activity.

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.

Temperament and attention: orienting toward and away from positive and negative signals.

Two studies used a target detection task to examine temperament-related attentional biases toward and away from significant stimuli. Pretarget cues were used to orient attention to locations carrying a positive incentive value (where points could be gained) or a negative value (where points could be lost). Under both involuntary and voluntary conditions, extraverts were slow to shift attention away from positive locations, whereas introverts were slow to shift from negative locations. These biases were enhanced on trials following negative feedback and tended to be strongest in Ss high in Neuroticism. The findings support models proposing that Extraversion reflects the combined activity of positive (strongest in extraverts) and negative (strongest in introverts) incentive motivational processes. They further suggest that incentive processes regulate the ability to shift attention away from, rather than toward, significant stimuli.

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA.

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.

Evaluation of immune parameters in HIV+ subjects reporting adverse reactions to sulfamethoxazole.

Trimethoprim-sulfamethoxazole (TMP-SMX) is frequently used in human immunodeficiency virus (HIV)-infected patients (HIV+) for treatment or prophylaxis of Pneumocystis carinii pneumonia (PCP). Up to 80% of those patients report adverse reactions to that drug combination. To test the hypothesis that these reactions are immunologically mediated, we quantitated specific IgG and IgE SMX-human serum albumin (HSA) antibodies and immune complexes (IC) in HIV+ patients and in HIV controls. Patients with mild HIV disease had elevated specific SMX-HSA IgG and IC levels compared with those having severe disease or with controls. Conversely, patients with severe HIV disease had statistically elevated levels of specific IgE when compared with patients having milder disease or with controls. There were no differences in either specific antibody or IC levels between patients reporting adverse reactions and those who did not. Results suggest that there are increased levels of SMX-HSA-specific antibodies in some HIV+ patients. The presence of these antibodies appears to be related to severity of disease, rather than clinically significant drug sensitivity.

Acidic mucin layer facilitates micelle dissociation and fatty acid diffusion.

The presence of a mucin layer on the surface of the intestinal epithelium has been suggested as an important factor in maintaining an acidic microclimate. The presence of such a low-pH compartment has been shown to facilitate fatty acid uptake. The mechanisms leading to the enhancement of fatty acid uptake were investigated in a purified acidic mucin layer. Our results indicate that the presence of a low-pH compartment indeed facilitates the dissociation of mixed micelles made of taurocholate and oleic acid. The released fatty acid formed an emulsion at the mucin layer, and this event could be visualized by the naked eye. When the size of the particles in the micelle solution was examined by photon correlation spectroscopy, it was found that acidification alone can lead to the formation of particles with size substantially greater than that of micelles. With the use of labeled fatty acid, the change in optical density can be correlated to the amount of fatty acid appearing in the mucin layer in an asymptotic fashion, suggesting that using the turbidity as an indicator might underestimate fatty acid diffusion. Despite this limitation, the rate of fatty acid diffusion in the mucin layer was estimated to be 400% of that in the buffer solution.

Evaluation of Basidiomycete and Deuteromycete (Fungi Imperfecti) extracts for shared allergenic determinants.

Aqueous extracts of select members of the Basidiomycetes and Deuteromycetes (Fungi Imperfecti) were evaluated for the presence of shared allergenic determinants using skin prick and radio-allergosorbent test (RAST) inhibition. Twenty adults with perennial symptoms of rhinitis, with or without asthma, were skin-prick tested with six species of Deuteromycetes and seven species of Basidomycetes. Positive weal-and-flare reactivity to Pleurotus ostreatus was associated with Alternaria alternata, Fusarium solani and Epicoccum purpurescens. Positive skin reactivity to Calvatia cyathiformis was also associated with A. alternata and F. solani. Coprinus quadrifidus was associated only with F. solani, and Psilocybe cubensis was only associated with Aspergillus fumigatus. No other skin test associations were demonstrated. For every allergen tested by RAST inhibition, significant dose-dependent homologous inhibition was demonstrated. Although the ability of an individual heterologous extract to inhibit the direct RAST varied, inhibition was generally minimal. In the most extreme example, no heterologous allergen inhibited the A. alternata RAST. However, the Armillaria tabescens RAST was inhibited 52.6%, 38.1% and 25.1% by A. fumigatus, E. purpurescens, and Penicillium notatum, respectively, suggesting significant cross-reactivity. These results suggest that, although shared allergenic determinants exist between select species of Basidiomycetes and Deuteromycetes, crossreactivity is minimal and its clinical significance is not clear. These data confirm that for reliable diagnosis of fungal allergy, representatives of both major groups must be used.

Imported fire ant (IFA) sensitivity: IFA-specific levels of serum IgG and IgG4.

Imported fire ant (IFA) whole body extract (WBE) and venom (V)-specific IgG and IgG4 antibodies and specific IgE antibodies were evaluated in sera from 56 IFA-sensitive individuals (18 undergoing immunotherapy with IFAWBE and 38 individuals not being treated) and 44 nonatopic and atopic control subjects with no history of IFA allergy. Although there was no difference in the level of IFAWBE- or IFAV-specific IgG between treated and untreated patients, both groups had higher levels of IFAWBE- and IFAV-specific IgG (p less than 0.05) than did control subjects. Patients receiving treatment tended to have higher levels of IFAWBE-specific IgG4 than did either untreated patients (0.05 less than p less than 0.10) or control subjects (p less than 0.05). Levels of IFAV-specific IgG4 were higher in treated patients than in control subjects (p less than 0.05) but were not different between treated and untreated patients. Levels of IFAWBE- and IFAV-specific IgE antibodies did not differ between the two patient populations but were higher in both groups than in control subjects (p less than 0.05). The ratio of IgG4 to IgE (G4/E) for both IFAWBE and IFAV was calculated for all groups. The ratios of IFAWBE- and IFAV-specific G4/E were higher in treated patients and in control subjects as compared to nontreated patients (p less than 0.05). IFAV-specific G4/E ratios were lower in treated patients than in control subjects, but IFAWBE-specific G4/E ratios were the same as ratios for control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts.

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.