Transient exciton-polariton dynamics in WSe2 by ultrafast near-field imaging.

Van der Waals (vdW) materials offer an exciting platform for strong light-matter interaction enabled by their polaritonic modes and the associated deep subwavelength light confinement. Semiconductor vdW materials such as WSe2 are of particular interest for photonic and quantum integrated technologies because they sustain visible-near-infrared (VIS-NIR) exciton-polariton (EP) modes at room temperature. Here, we develop a unique spatiotemporal imaging technique at the femtosecond-nanometric scale and observe the EP dynamics in WSe2 waveguides. Our method, based on a novel ultrafast broadband intrapulse pump-probe near-field imaging, allows direct visualization of EP formation and propagation in WSe2 showing, at room temperature, ultraslow EP with a group velocity of v g ~ 0.017c. Our imaging method paves the way for in situ ultrafast coherent control and extreme spatiotemporal imaging of condensed matter.

Function-modulating human monoclonal antibodies against platelet-membrane receptors isolated from a phage-display library.

Monoclonal antibodies to platelet membrane receptors have been used extensively for analysis of receptor structure and function. Function-blocking human antibodies are being used for the development of antiplatelet drugs. We isolated human monoclonal antibodies from a library of single-chain Fv (scFv) antibodies displayed on the surface of filamentous phage, by selection on whole platelets. Eight different platelet-binding clones were isolated, of which three bound to the platelet-membrane glycoprotein (GP) GPIb in an ELISA assay. Specific elution with a recombinant polypeptide of von Willebrand factor (VWF) spanning the GPIbalpha binding site, yielded the same three phage clones. Two of the three anti-GPIb clones could be purified as scFv monoclonal antibodies, and they competed with each other for binding to intact platelets, suggesting that they bind at or near the same site on GPIb. Their binding affinities differed, however, and the clone with higher affinity inhibited ristocetin-induced platelet aggregation. These data indicate that selection from a phage display library of human scFvs using whole platelets can be applied for the isolation of functional antiplatelet-GPIb antibodies useful for the development of new therapeutic and diagnostic strategies.

[Hemorrhoidectomy using a circular stapler].

We compared traditional local excision of hemorrhoids in 40 patients (group A) with excision using the CDH-33 surgical stapler designed for bowel anastomosis, in 41 (group B). In this technique a purse-string suture is prepared 3 cm above the dentate line, and the stapler is inserted and used to form a mucosa-mucosal anastomosis. This technique is less time-consuming than the traditional technique. Data were collected from patients' medical files and from detailed questionnaires in which symptoms prior to and after operation were reported. Mean ages were 53.0 and 51.5 years and male/female ratios were 1.0:1.2 and 1.0:1.1 respectively, neither significant. The most common complaints in both groups were pain and rectal bleeding. All patients had a lower-GI investigation prior to operation to exclude other causes of rectal bleeding. Recovery averaged 2 months in both groups. Patient satisfaction was assessed by decrease or absence of symptoms on return to normal daily activities. Satisfaction tended to be greater in group B. More patients in group A complained of tightness and discomfort at the operative site, but this was not significant. We are extending our study to a larger number of patients to determine if there are statistically significant differences between the results of the 2 methods.

Sleep characteristics in children with Down syndrome.

BACKGROUND: Obstructive sleep apnea syndrome is common in children with Down syndrome (DS). Little is known about sleep patterns, especially arousals, awakenings, and movements during sleep in children with DS. OBJECTIVE: To determine the characteristics of sleep disorders in children with DS and to define the associations between respiratory disturbance and arousals, awakenings, and movements. METHODS: The study included 23 children with DS, compared with 13 children with primary snoring. All underwent a 6- to 8-hour sleep study. RESULTS: The respiratory disturbance index was significantly higher in the children with DS (2.8 +/- 2.3 events/h vs 0.6 +/- 0.4 events/h; P <.05). Sleep was significantly fragmented in children with DS, who had a significantly higher arousal/awakening (A/Aw) index (24.6 +/- 7.9 events/h) compared with the comparison group (17.6 +/- 4.0 events/h) (P <.02). A higher percentage of jerks associated with A/Aw and respiratory event-associated A/Aw was observed in patients with DS (45.2% +/- 25% and 8.6% +/- 6.4%, respectively) compared with the control patients (10.2% +/- 4.5% and 1.5% +/- 2.1%) (P <.02). The median length of occurrences of stage 2 sleep was 27% shorter in the DS group (P <.03). The number of shifts from "deeper" to "lighter" stages of non-rapid eye movement sleep was 30% greater (P <.02) in the DS group. CONCLUSION: Children with DS have significant sleep fragmentation, manifested by frequent awakenings and arousals, which are only partially related to obstructive sleep apnea syndrome.

Recombinant polypeptides derived from the fibrin binding domain of fibronectin are potential agents for the imaging of blood clots.

Thrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa ("2 fingers"), 18.5 kDa ("3 fingers") and 31 kDa FBD ("5 fingers"), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C-putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111In]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [111In]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

Direct observation of soliton and normal pulse propagation by spectral domain reflectometry.

We report what are to our knowledge the first continuous observations of optical fiber solitons and nonsoliton pulses in the spectral domain. Using a novel optical time-resolved spectral ref lectometer that measures the backscattered light spectrum, we directly observed (with 200-m spatial resolution) the propagation of ~8.5-ps solitons and nonsolitons traveling down 5-km fibers. Spectral breathing and the recovery of the original spectral widths are clearly seen for higher-order solitons. Possible applications include in situ measurements of soliton parameters, fiber dispersions, and nonlinearities, experimental verifications of pulse propagation theories, and optical sensing.

The collagen binding domain of fibronectin contains a high affinity binding site for Candida albicans.

A 30-kDa proteolytic fragment from the gelatin/collagen-binding domain of fibronectin is a potent inhibitor of fibronectin binding to Candida albicans, with a molar inhibition constant equal to that of intact fibronectin. Recombinant and proteolytic fragments from the cell-, the fibrin I-, and the heparin II-binding domains also inhibit fibronectin binding, but are 13-1000-fold less active. In suspension, binding of fibronectin to C. albicans is regulated by growth conditions and is specific, saturable, time-dependent, reversible, and divalent cation-independent. Scatchard plot analyses indicate the presence of high affinity (Kd = 1.3 x 10(-9) M) and low affinity (Kd = 1.2 x 10(-7) M) receptors. Recombinant or proteolytic fragments from four binding domains of fibronectin promote adhesion of C. albicans. A recombinant fragment corresponding to the cell-binding domain but with the sequence Arg-Gly-Asp-Ser deleted promotes C. albicans adhesion and inhibits fibronectin binding to C. albicans with the same activity as the natural sequence. Furthermore, four peptides containing the Arg-Gly-Asp-Val sequence and the peptides CS-1 and Arg-Glu-Asp-Val did not block the binding of fibronectin to C. albicans. Thus, in contrast to the specific binding of soluble fibronectin, recognition of immobilized fibronectin by C. albicans is mediated by several domains of the protein. Interactions with the cell-binding domain are not mediated by the Arg-Gly-Asp or other known recognition sequences as it has been suggested. Binding of fibronectin also did not correlate with C3d binding to the avirulent clones of C. albicans strain H12 or with iC3b binding to variants of the strain 4918.

Studies on fibronectin and its domains. I. Novel recombinant cell-binding domain of fibronectin--a modulator of human platelet functions.

The DNA sequences encoding for two proteins of the cell-binding domain (CBD) of human fibronectin (FN), namely a 33-kDa protein (aa 1329-1722) and a 40-kDa (aa 1380-1851) protein, were cloned and expressed in Escherichia coli. The interactions of the resulting rCBD proteins, refolded and purified to homogeneity, with human platelets were studied in comparison with those of the pentapeptide GRGDS. The binding of both the 33-kDa and the 40-kDa proteins to washed platelets appeared to be dependent upon platelet activation. In the case of the 33-kDa protein, binding to stimulated platelets was shown to be saturable, with Kd = 2 microM (thrombin as agonist). Moreover, both the 33-kDa and the 40-kDa proteins inhibited fibrinogen binding (at 0.1 microM) to ADP- or thrombin-stimulated platelets with IC50 values in the same concentration range. Binding seemed therefore to occur mainly at the GPIIb/IIIa receptor, and accordingly monoclonal antibodies against this receptor prevented up to 85% of the binding of the 33-kDa protein to platelets. With most stimuli the 33-kDa and the 40-kDa proteins inhibited platelet aggregation at concentrations 15- to 25-fold lower than those required by GRGDS and, in the case of the 33-kDa protein, this was shown to occur in either platelet-rich plasma, washed platelets, or whole blood. The 33-kDa protein also inhibited platelet aggregation and thromboxane A2 (TXA2) generation on the subendothelial extracellular matrix, whereas the GRGDS peptide inhibited only matrix-induced platelet aggregation, but not TXA2 formation. Furthermore, the 33-kDa protein, which is derived from the human FN CBD, seemed to be highly selective, since it inhibited the aggregation of platelets from primates only, and not from other animals tested. Finally, the 33-kDa protein did not promote fibroblast cell attachment, as was observed for both whole FN and the 40-kDa protein, thus displaying a selectivity toward platelets. In conclusion, the unique properties of the 33-kDa protein, and, in particular, its special affinity directed only toward activated primate platelets, seem to hold a promising potential for the further development of an antithrombotic agent.

Recombinant carp (Cyprinus carpio) growth hormone: expression, purification, and determination of biological activity in vitro and in vivo.

Carp growth hormone (cGH) cDNA (Koren et al., 1989) was cloned under the control of lambda-phage PLOL promoter and lambda cll ribosomal binding site into pBR322 plasmid to enable its expression in Escherichia coli A1645 that produces constitutively the thermolabile lambda repressor c1857. Temperature shift to 42 degrees abolished the repression, resulting in a high level of cGH expression. The bacterially expressed cGH protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on Q-Sepharose column by stepwise elution with NaCl. The bioactive fraction was eluted at 0.2 M NaCl at a yield of 10-15%. This fraction contained predominantly (95%) 21.5-kDa monomeric cGH. The activity of cGH in vitro was bioassayed using Nb2-11C lymphoma cells (containing lactogenic receptors) and 3T3-F442A preadipocyte cells (containing somatogenic receptors). Bioactivity was found to be 0.01 and 6-10% that of human GH, respectively. In vivo cGH activity was measured by weekly ip injection in juvenile carp fed a low (23%) protein diet. Over a 6-week period, cGH increased the growth rate by 38% compared to fish injected with vehicle only. Identical injections with bovine GH yielded only a 21% increase.

Preparation and comparison of biological properties of recombinant carp (Cyprinus carpio) growth hormone and its Cys-123 to Ala mutant.

Carp growth hormone (cGH) cDNA, in which Cys-123 was mutated to Ala, was prepared, transferred to the expression vector, expressed in Escherichia coli and the mutant was purified to homogeneity. The mutation only slightly improved yield of the monomeric fraction, indicating that Cys-123 is not involved in improper refolding. As compared to cGH, the mutant (cGH-C123A) exhibited lower binding affinity toward homologous liver receptors and lower bioactivity in a 3T3-F442A preadipocyte bioassay despite the fact that both hormones exhibited almost identical cross-reactivity with anti-cGH antibodies. These results, along with those of a structural comparison to hGH, suggest that Cys-123 is located in the hydrophobic core of the hormone, and is most likely affecting the conformation of the binding site. Dimeric forms of the hormone and its mutant were less active than their respective monomers. Homologous binding experiments using a carp liver microsomal fraction revealed a single receptor population with Kd = 0.77 nM and Bmax = 241 fmol/mg microsomal protein.

Selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in Escherichia coli.

The maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. In this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 cI- prophage. Cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. The presence of the phage 434 repressor in the cells does not interfere with the process of lambda repressor inactivation and the high-level production of bovine growth hormone.

Site-specific mutagenesis of human apolipoprotein E. Receptor binding activity of variants with single amino acid substitutions.

Apolipoprotein (apo) E, an important protein involved in cholesterol transport in the plasma, binds with high specificity and high affinity to the apoB, E (low density lipoprotein) receptor. Several lines of evidence have indicated that key basic residues in the vicinity of residues 140-160 of apoE are important in mediating binding to the receptor. Furthermore, apoE variants exhibiting defective receptor binding are associated with the genetic lipid disorder type III hyperlipoproteinemia. To determine whether other basic amino acids in this region of apoE also affect receptor binding activity, site-specific mutagenesis of apoE in a bacterial expression system was undertaken. This system had been used successfully to produce apoE3 that was structurally and functionally equivalent to human plasma apoE3. Variants of apoE in which neutral amino acids were substituted for basic residues at positions 136, 140, 143, and 150 were produced. The variants all displayed defective binding; their activity ranged from 9 to 52% of normal (a range similar to that seen with naturally occurring variants of human apoE). In addition, to determine whether the conformation of this region is important for receptor binding, we designed variants in which proline was substituted for leucine 144 or alanine 152. Both variants were defective, exhibiting 13 and 27% of normal binding, respectively. In contrast, a double mutant in which arginine was substituted for serine 139 and alanine for leucine 149 displayed slightly enhanced receptor binding activity. These studies confirm that the middle of the apoE molecule is important in receptor binding and indicate that only certain amino acid substitutions in this region interfere with receptor binding activity.

Comparative study of in vitro and in vivo modulation of lactogenic and somatotropic receptors by native human growth hormone and its modified analog prepared by recombinant deoxyribonucleic acid technology.

A modified analog of human GH (hGH), prepared by recombinant DNA technology, that lacks 13 amino acids at the amino terminus (Met14hGH), was able to compete with [125I]hGH for binding to lactogenic receptors in Nb2-11C rat lymphoma cells, to somatotropic receptors in IM-9 human lymphocytes, and to both lactogenic and somatotropic receptors in the microsomal fraction of virgin female rat liver. Exposure of intact Nb2 or IM-9 cells to Met14hGH did not reduce the number of surface or intracellular receptors, as compared to the control without hormone. A parallel exposure to 500-fold lower concentrations of hGH resulted in 77-93% reduction in both surface and intracellular receptors. In contrast to [125I]hGH, [125I]Met14hGH was not taken up by the intact Nb2 lymphoma cells. Infusion of anesthetized female virgin rats for 3 h with hGH down-regulated both lactogenic and somatotropic receptors in the liver. A similar infusion with up to 200-fold higher amounts of Met14hGH did not lower the number of total receptors, indicating lack of down-regulation. Some decrease in the binding to free receptors was observed, suggesting that Met14hGH is capable of binding to liver receptors in vivo.

Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of a modified recombinant hGH.

A recombinant analog of human GH (hGH) lacking 13 amino acids at the amino-terminus (Met14hGH) inhibited the hGH- or ovine PRL (oPRL)-stimulated proliferation of Nb2 lymphoma cells and bovine PRL-stimulated fat synthesis and alpha-lactalbumin secretion in explants from bovine lactating mammary gland. The inhibition was competitive in nature, and in Nb2 cells could be abolished by an excess of hGH or oPRL. Inhibition of oPRL-stimulated proliferation of Nb2 cells by Met14hGH could also be specifically abolished by anti-hGH monoclonal antibodies. Met14hGH had no growth-stimulating activity in Nb2 cells and was not cytotoxic. It also did not affect glucose uptake by the mammary gland explants. Met14hGH competed with [125I]hGH for binding to intact Nb2 cells, IM-9 lymphocytes, solubilized microsomal fraction from lactating bovine mammary gland, and microsomal fraction from the liver of female virgin rats, but its affinity for those receptors was 2 orders of magnitude lower than the affinity of hGH. Since Met14hGH used in most experiments contained about 25% impurities and degradation products, a small amount of it was further purified by immunoaffinity chromatography. Two purified fractions, one consisting of a single 20K protein and the other accompanied by a small amount of 25K protein, were obtained. Both fractions exhibited increased inhibition of hGH- or oPRL-stimulated proliferation of Nb2 cells, thus indicating that the inhibitory activity results from the intact Met14hGH molecule. To the best of our knowledge, this is the first report describing the inhibition of lactogenic hormone activities by a modified hGH.

Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.

Fluorescence polarization of DPH-labeled cells adsorbing viruses and its diagnostic potential.

Mammalian or avian cells were labeled with a fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene). Within a few minutes after adsorption of various naked and enveloped viruses, the degree of fluorescence polarization (P) of the DPH embedded in the adsorbing cells as measured at 37 degrees C, was reduced, a finding indicating a decrease in the microviscosity of the lipids in the cell membrane. This change of fluidity was proportional to the concentration of the adsorbing virus and could be abolished or inhibited by homologous specific antiviral sera, but not by heterologous sera. Potential use of fluorescence polarization tests is described for titration of virus concentration, as well as for serological identification of a virus.