Quantitative modeling of near-field interactions incorporating polaritonic and electrostatic effects.

As scattering-scanning near-field optical microscopy (s-SNOM) continues to grow in prominence, there has been great interest in modeling the near-field light-matter interaction to better predict experimental results. Both analytical and numerical models have been developed to describe the near-field response, but thus far models have not incorporated the full range of phenomena accessible. Here, we present a finite element model (FEM), capable of incorporating the complex physical and spatial phenomena that s-SNOM has proved able to probe. First, we use electromagnetic FEM to simulate the multipolar response of the tip and illustrate the impact of strong coupling on signal demodulation. We then leverage the multiphysics advantage of FEM to study the electrostatic effect of metallic tips on semiconductors, finding that THz s-SNOM studies are most impacted by this tip-induced band-bending. Our model is computationally inexpensive and can be tailored to specific nanostructured systems and geometries of interest.

Response of Arthrospira platensis to nitrogen depletion and the effect of aqueous extracts on tumor and non-tumor cells.

The microalgae Arthrospira platensis (AP), commonly known as Spirulina, has gained widespread popularity as a food supplement in recent years. AP is particularly abundant in protein, B vitamins, iron, magnesium, potassium, and various antioxidants. In this study we aimed to evaluate the effect of nitrate limitation in the AP culture medium on AP growth and composition. In addition, the cytotoxicity of the respective aqueous AP extracts on three different mammalian cell-lines (HepG2, Caco2, L929) was tested. AP was cultivated over a 10-day period under nitrogen-rich (Nrich: 1.8 g/L) and nitrogen-deficient (Nlimited: 0.2-0.4 g/L) conditions in two separate experiments, each with three biological replicates (three bioreactors). Throughout the cultivation, the kinetic progress of dry biomass, pH, pigment content, the levels of essential elements (sulphur, phosphate, and nitrate) and the composition of elements in the harvested biomass was determined. While the biomass slightly but significantly differed, the phycocyanin concentration differed considerably (around 10-fold higher in the Nrich medium, p < 0.05). Aqueous extracts of the Nrich medium had significantly stronger effects on the cell membrane integrity and the metabolic activity of the cells than extracts of the Nlimited medium. Particularly was the finding that AP had a significantly stronger toxic effect on the two tumour cell types (HepG2, Caco2) than on the non-tumour cells (L929). This study underscores the significance of nitrate content in the cultivation media of AP.

Onset of sliding in amorphous films triggered by high-frequency oscillatory shear.

We investigate the change of the static friction threshold of weakly adhesive amorphous interfaces in the presence of the shear ultrasonic oscillation. Prior to sliding, a softening of the shear interfacial stiffness is observed under either static or high-amplitude oscillatory shear. We find that the nonlinear shear ultrasound, regardless of its polarization, triggers the macroscopic sliding at these interfaces far below the static threshold. Such unjamming transition is due to the vibration-induced decrease of the apparent coefficient of static friction, which provides a mechanism for understanding the reduction of the yielding threshold of granular media by the acoustic fluidization.

Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis.

Lizards cannot naturally regenerate limbs but are the closest known relatives of mammals capable of epimorphic tail regrowth. However, the mechanisms regulating lizard blastema formation and chondrogenesis remain unclear. Here, single-cell RNA sequencing analysis of regenerating lizard tails identifies fibroblast and phagocyte populations linked to cartilage formation. Pseudotime trajectory analyses suggest spp1+-activated fibroblasts as blastema cell sources, with subsets exhibiting sulf1 expression and chondrogenic potential. Tail blastema, but not limb, fibroblasts express sulf1 and form cartilage under Hedgehog signaling regulation. Depletion of phagocytes inhibits blastema formation, but treatment with pericytic phagocyte-conditioned media rescues blastema chondrogenesis and cartilage formation in amputated limbs. The results indicate a hierarchy of phagocyte-induced fibroblast gene activations during lizard blastema formation, culminating in sulf1+ pro-chondrogenic populations singularly responsive to Hedgehog signaling. These properties distinguish lizard blastema cells from homeostatic and injury-stimulated fibroblasts and indicate potential actionable targets for inducing regeneration in other species, including humans.

Synthetic gene circuits as tools for drug discovery.

Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays.

Platelet activating factor raises intracellular calcium ion concentration in macrophages.

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.

The role of extracellular materials in cell movement. I. Inhibition of mucopolysaccharide synthesis does not stop ruffling membrane activity or cell movement.

The involvement of mucopolysaccharide synthesis in cell locomotion was investigated by determining the effects of inhibition of synthesis on ruffling membrane activity and cell movement by embryonic heart fibroblasts. Mucopolysaccharide synthesis was inhibited directly by treatment with a glutamine analog, 6-diazo-5-OXO-L-norleucine (DON), and indirectly with cycloheximide. DON treatment reduced synthesis to 20% of control values, and cycloheximide reduced synthesis to less than 10% of control values, as measured by incorporation of [35S]sulfate into mucopolysaccharides. Nevertheless, ruffling membrane activity and cell locomotion continued under both conditions. Cytochalasin B did not inhibit mucopolysaccharide synthesis, although it did stop ruffling and locomotion. These results suggest that if mucopolysaccharides are required for cell movement, they must have long half-lives or represent only a minute fraction of the normal synthetic load.

A model for astral stimulation of cytokinesis in animal cells.

A model is proposed in which stimulation of cortical cytoplasm occurs near the distal ends of astral rays. Levels of stimulation sufficient to cause furrowing occur only in equatorial zones between asters. The model can account for positioning of furrows in very large cells (fertilized eggs of amphibians, birds, and fish) and in cells with several mitotic apparatuses (insects). Finally, the model correctly predicts the positioning and occurrence of furrowing in two experiments in which cellular shape was manipulated into either an hourglass or a cylindrical form before division. These results are consistent with equatorial stimulation theories in which mitotic asters differentially stimulate the future furrow region (equatorial cortex). The results are not consistent with models requiring differential stimulation of nonfurrowing, polar regions of the cell.

Synthesis of type III collagen by fibroblasts from the embryonic chick cornea.

Synthesis of collagen types I, II, III, and IV in cells from the embryonic chick cornea was studied using specific antibodies and immunofluorescence. Synthesis of radioactively labeled collagen types I and III was followed by fluorographic detection of cyanogen bromide peptides on polyacrylamide slab gels and by carboxymethylcellulose chromatography followed by disc gel electrophoresis. Type III collagen had been detected previously by indirect immunofluorescence in the corneal epithelial cells at Hamburger-Hamilton stages 20--30 but not in the stroma at any age. Intact corneas from embryos older than stage 30 contain and synthesize type I collagen but no detectable type III collagen. However, whole stromata subjected to collagenase treatment and scraping (to remove epithelium and endothelium) and stromal fibroblasts from such corneas inoculated in vitro begin synthesis of type III collagen within a few hours while continuing to synthesize type I collagen. As demonstrated by double-antibody staining, most corneal fibroblasts contain collagen types I and III simultaneously. Collagen type III was identified biochemically in cell layers and media after chromatography on carboxymethylcellulose be detection of disulfide-linked alpha l (III)3 by SDS gel electrophoresis. The conditions under which the corneal fibroblasts gain the ability to synthesize type III collagen are the same as those under which they lose the ability to synthesize the specific proteoglycan of the cornea: the presence of corneal-type keratan sulfate.

Creatine kinase release, potassium-42 content, and mechanical performance in anoxic rabbit myocardium.

We studied whether creatine kinase appearance in venous effluent was specific for, and quantitatively proportional to, the amount of loss of functioning myocardium. Cell viability was determined by simultaneously monitoring tissue (42)K content and mechanical performance during anoxia and reoxygenation in isolated, arterially perfused, interventricular rabbit septa. The septa were paced at 42 beats/min and perfused at 1.8 ml/min per g tissue with a modified Tyrode solution at 28 degrees C. Net total creatine kinase losses of 5.3+/-2.7, 20.6+/-7.2, 55.3+/-7.6, and 110.7+/-27.1 IU/g dry wt (mean+/-SEM) were observed after 20, 30, 40, and 60 min of anoxia, respectively. Maximum (42)K losses during the same intervals of anoxia were 16.8+/-3.4, 38.3+/-2.9, 47.0+/-1.4, and 84.3+/-14.8 mmol K(+)/kg dry wt and correlated with creatine kinase losses, r = 0.97. Upon reoxygenation, (42)K content returned to a new plateau which was expressed as a percentage of decrease from control content. These unrecovered (42)K losses were -2.7+/-0.9, 0.7+/-2.9, 6.6+/-1.9, and 14.0+/-6.5% after 20, 30, 40, and 60 min of anoxia, respectively, and correlated with the creatine kinase loss, r = 0.97. Net loss of developed tension after reoxygenation was 9.0+/-2.3, 26.7+/-17.9, 31.7+/-1.1, and 60.7+/-8.8% of control after these anoxic intervals and correlated with creatine kinase loss, r = 0.92. The small enzyme loss that occurred after 20 min anoxia without evidence for irreversible loss of cell function was congruent with0.1% of total tissue enzyme content. The significant correlation of enzyme loss with the irreversible losses of potassium content and contractile performance supported the hypothesis that creatine kinase appearance in the venous effluent was the result of cell death.

Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site.

Mimecan is a small leucine-rich proteoglycan that can occur as either keratan sulfate proteoglycan in the cornea or as glycoprotein in many connective tissues. As yet, there is no information on its transcriptional regulation. Recently we demonstrated the presence of eight mimecan mRNA transcripts generated by alternative transcription initiation, alternative polyadenylation, and differential splicing, all of which encode an identical protein. Here we report a conserved consensus p53-binding DNA sequence in the first intron of bovine and human mimecan genes and show that wild-type p53 binds to this sequence in vitro. Co-transfections of Saos-2, HeLa, NIH 3T3, and primary bovine corneal keratocytes with bovine mimecan promoter/luciferase reporter constructs in combination with p53 expression vectors activate the second mimecan promoter through the p53-binding sequence. In addition, we show absence of mimecan expression in different tumors and cancer cell lines, where p53 frequently is inactivated/mutated. Thus, this work provides novel information that links mimecan to the p53 network.

Identification and characterization of conserved cis-regulatory elements in the human keratocan gene promoter.

Keratocan, along with lumican and mimecan, represent the keratan sulfate-containing proteoglycans of the vertebrate cornea that play a key role in development and maintenance of corneal transparency. In this study, we cloned 4.1 kb of the human Kera 5'-flanking region and characterized the promoter structure. Using primer extension and ribonuclease protection assay, we identify two major transcriptional start sites in the first exon. Using luciferase reporter gene transfection analysis of 5'-deletion and mutation constructs, we demonstrate positive and negative regulatory elements within a 1.3 kb upstream sequence. Comparison of human and bovine 5'-flanking sequences reveals three highly conserved regions: a 450 bp region in the first exon, a 92 bp promoter proximal conserved regulatory region identified as an enhancer in the natural context, and a 223 bp promoter distal conserved regulatory region identified as a silencer both in the natural context and in a heterologous promoter system. In addition, a conserved CArG-box residing 851 bp upstream of the first transcription start site also can lead to the repression of Kera expression in cultured corneal keratocytes. DNaseI footprinting and electrophoretic mobility shift assay demonstrate that cell type-specific factors bind to regulatory elements located in the conserved regions. Competition experiments indicate that the CTC factor and a protein that binds to the CAGA motif are likely to be among the multiple factors involved in the transcriptional regulation of the human Kera gene.

Indium-111 platelet scintigraphy and two-dimensional echocardiography for detection of left ventricular thrombus: influence of clot size and age.

Two-dimensional echocardiography and indium-111 platelet scintigraphy were performed on 50 dogs to determine the influence of clot age and size on the detection of experimentally induced left ventricular mural thrombus. Thrombus was induced by apical infarction and injection of a sclerosing agent and thrombin. The animals were classified into four groups according to the time of indium-111 platelet injection after thrombus induction: Group I (17 dogs, 1/2 hour after induction; 3 dogs, before induction), Group II (12 dogs, 24 hours after induction) and Group III (12 dogs, 1 week after induction). In Group IV (six control dogs) apical infarction was produced, but thrombin was not injected; indium-111 platelets were injected 1/2 to 1 hour after infarction. The dogs were studied by indium-111 platelet scintigraphy and by two-dimensional echocardiography 1/2 to 5 hours (Group I) and 1 to 5 and up to 72 hours (Groups II to IV) after platelet administration and before death was induced. Two-dimensional echocardiography showed the best overall sensitivity for detection of acute thrombus (97%; 29 of 30). The sensitivity of indium-111 platelet scintigraphy was 86% (18 of 21) for clots greater than or equal to 0.08 ml in size, and 67% (20 of 30) for detection of all clots. Thrombus did not form in 14 dogs of Groups I to III and in 6 of 6 control dogs. The specificity of scintigraphy was 100% (20 of 20) compared with 80% (16 of 20) for echocardiography. Echocardiography was more sensitive than scintigraphy for detecting very small clots in this experimental model.(ABSTRACT TRUNCATED AT 250 WORDS)

Perturbation of the yield-stress rheology of polymer thin films by nonlinear shear ultrasound.

We investigate the nonlinear response of macromolecular thin films subjected to high-amplitude ultrasonic shear oscillation using a sphere-plane contact geometry. At a film thickness comparable to the radius of gyration, we observe the rheological properties intermediate between bulk and boundary nonlinear regimes. As the driving amplitude is increased, these films progressively exhibit oscillatory linear, microslip, and full slip regimes, which can be explained by the modified Coulomb friction law. At highest oscillation amplitudes, the interfacial adhesive failure takes place, being accompanied by a dewettinglike pattern. Moreover, the steady state sliding is investigated in thicker films with imposed shear stresses beyond the yield point. We find that applying high-amplitude shear ultrasound affects not only the yielding threshold but also the sliding velocity at a given shear load. A possible mechanism for the latter effect is discussed.

Molecular cloning and relative tissue expression of keratocan and mimecan in embryonic quail cornea.

We have cloned and sequenced the cDNAs for quail cornea keratan sulfate proteoglycan core proteins, keratocan and mimecan. The deduced quail keratocan protein contains a single conservative amino acid difference from the chick sequence, whereas quail mimecan protein contains a 58 amino acid-long avian-unique sequence that shares no homology with mammalian mimecan. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that keratocan and lumican are expressed at highest levels in cornea, whereas mimecan mRNA is expressed at a much lower level. Keratocan is expressed only in quail cornea, whereas mimecan is expressed in many different tissues as four transcripts of different sizes. Both lumican and mimecan are expressed at lowest levels in brain, liver and sternum.

Molecular cloning and relative tissue expression of decorin and lumican in embryonic quail cornea.

We have cloned and sequenced the cDNAs for quail cornea proteoglycan core proteins, decorin and lumican. Comparison of deduced amino acid sequences shows that two of five amino acid differences in the mature protein between quail and chick decorin, and two of three for lumican, are non-conservative. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that decorin and lumican are most highly expressed in cornea, and that both are also highly expressed at approximately equal levels in most other tissues. Decorin is highly expressed in sclera and sternum, whereas lumican is expressed in these tissues, as well as in liver, at very low levels. Both decorin and lumican are expressed at lowest levels in brain.

Cloning, characterization and tissue-specific expression of the gene encoding bovine keratocan, a corneal keratan sulfate proteoglycan.

Keratocan is one of three major keratan sulfate proteoglycans characteristically expressed in cornea. We reported previously the sequence of bovine Kera cDNA. In this study, the complete bovine Kera gene was cloned and sequenced, and its expression pattern was determined. The Kera gene is composed of three exons and two introns that span 8.830kb of the bovine genome. The first exon contains 287 nucleotides of 5'-UTR sequence. Both of the two large introns of 1322 and 4178bp contain (CA)n repeats. The bovine Kera gene has a TATA box that is located 28bp upstream from tsp. Primer extension and S1 nuclease protection analyses were used to determine the major tsp. RPA indicate that cornea and sclera are the two tissues with the highest expression of Ktcn mRNA. This restricted expression in eye tissues, as well as the unique modification of keratocan with long keratan sulfate chains in cornea, suggests that this molecule may be important in developing and maintaining corneal transparency.

Renal arteriovenous transit times of technetium-radiolabeled chelates.

A noninvasive method was developed for quantitating the distribution of renal arteriovenous transit times of technetium-99m (99mTc) radiopharmaceuticals. Using this method, the characteristic transit times and amplitudes of the first two components of [99mTC] DTPA or MDP transit through the renal vasculature were calculated. The first component amplitude (A1) was evaluated for its ability to discriminate between 20 hypertensive patients with renovascular disease and 21 normotensive subjects. A1 was compared with three other quantitative indices: the ascending slope of the initial renal time-activity curve, the kidney-to-aorta slope ratio (K/A), and renal size. A1 nearly perfectly separated the hypertensive patients from the normotensive subjects; the ability of A1 to discriminate between these two groups is clearly superior to renal size, the initial renal slope, and K/A. We conclude that measurements of the intrarenal distribution of blood flow have distinct advantages over indices of renal blood flow that have been derived from scintillation camera measurements of 99mTc radiopharmaceuticals.

Pitfalls in establishing the diagnosis of deep venous thrombophlebitis by indium-111 platelet scintigraphy.

Forty-seven 111In-platelet scintigraphs (In-PS) were analyzed retrospectively to identify sources of diagnostic error and to optimize the diagnostic criteria for active deep venous thrombophlebitis (DVT). The results of In-PS were compared with contrast venography, additional diagnostic studies, and clinical outcome. Three patterns of platelet localization emerged as the best predictors of active DVT: (a) focal or (b) linear 4-hr localization, or (c) an asymmetric blood-pool pattern on 4-hr imaging that evolved into a focal or linear pattern by 16 to 24 hr. All false-positive studies had abnormal patterns confined to the inguinal region at 24 hr. All patients with false-negative studies had received heparin between 4 and 24 hr. The potential pitfalls encountered in the evaluation of the iliac, femoral, and popliteal veins are reviewed and the importance of delayed imaging in selected cases is emphasized.

Acetylcholinesterase activity in the cornea of the developing chick embryo.

Activity of acetylcholinesterase (EC 3.1.1.7) and pseudocholinesterase (EC 3.1.1.8) was measured in extracts from chick corneas, in a developmental series from days 7-20 of incubation and at three ages after hatching. Enzyme activity was measured by the biphasic single-vial radiometric assay of Johnson and Russell using [3H-acetyl]choline as substrate. Pseudocholinesterase was inhibited with tetraisopropylpyrophosphoramide (iso-OMPA). True acetylcholinesterase activity was verified by control assays run in the presence of both iso-OMPA and the true acetylcholinesterase inhibitor, 1:5-bis(4-allyldimethyl-ammonium phenyl)-pentane-3-one diiodide (BW284c51). With both inhibitors present, no cholinesterase activity was detected. Corneal acetylcholinesterase had an average Km of 1.1 +/- 0.3 X 10(-3) M at day 7, 14, and 20 of development and retained 90% activity even after 3 hr at 26 degrees C. At least 90% of the total cholinesterase activity was solubilized by Triton X-100 and sonication treatment. Activity decreased with increasing concentrations of NaCl present in the assay. A 60-fold transient increase in acetylcholinesterase specific activity occurs during the period from days 7-20 of embryonic development. This increase begins on the first day measured (day 7), progresses steadily and rapidly during the subsequent week, reaches a peak at day 15, and then decreases rapidly before hatching to a level maintained into adulthood. A similar pattern of transient appearance of highly sialylated gangliosides seen previously on days 14-17 leads to an hypothesis of a structural linkage between acetylcholinesterase and the plasma membrane lipids of corneal epithelial cells.