Time-dependent responses of wounded human skin fibroblasts following phototherapy

BACKGROUND AND OBJECTIVE: The penetration and distribution of laser light in target tissue is dependent on the wavelength of the light. One problem with most of the published data on laser irradiation is that most studies do not record the duration between the exposure and the evaluation. This study aimed to establish if the dose, wavelength or duration of effect (1h or 24h) influences the biological responses of irradiated fibroblasts. MATERIALS AND METHODS: The study established cellular responses of normal and wounded human skin fibroblasts to helium-neon (632.8 nm), diode (830 nm) and Nd:YAG (1064 nm) laser irradiation using one exposure of 5 J/cm(2) or 16 J/cm(2) on day 1 and again on day 4. Cellular responses to laser irradiation were evaluated by measuring changes in cell viability (ATP viability and caspase 3/7 activity) and cell proliferation (ALP enzyme activity and bFGF expression), 1h and 24h post irradiation. RESULTS: Wounded cells exposed to 5 J/cm(2) using 632.8 nm showed an increase in ATP viability after 1h, a decrease in caspase 3/7 activity after 24h and an increase in cell proliferation after 24h. The results suggest that changes in parameters such as ATP viability should be observed directly after laser irradiation (1h) whereas other parameters such as caspase 3/7 activity, bFGF expression and ALP enzyme activity should be measured at least 24h after the final exposure. CONCLUSION: This study confirms that the duration of effect should be included as one of the main laser parameters when reporting on the effects of laser irradiation. It is important to establish time-dependent responses as the results may provide an understanding of the cellular responses following laser irradiation.     

The effect of smoothing filter slope and spectral frequency on temporal speech information

It is known that information contained within the filter skirts can provide cues important to speech intelligibility. However, the role of filter slope during temporal smoothing has received little attention. In experiment 1, smoothing filter slope angle was found to have a large effect on the intelligibility of sentences represented by three amplitude-modulated sinusoids. In experiment 2, the use of temporal cues above 16 Hz was examined across various regions of the spectrum. When increases in rate were presented to individual spectral bands, intelligibility only increased when presented in the higher spectral region. This result suggests a greater reliance on higher-rate cues in this region. However, intelligibility was greatest when these cues were distributed across the spectrum, indicating that their effective use is not restricted solely to this region.     

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

Controlled loading of cryoprotectants (CPAs) to oocyte with linear and complex CPA profiles on a microfluidic platform

Oocyte cryopreservation has become an essential tool in the treatment of infertility by preserving oocytes for women undergoing chemotherapy. However, despite recent advances, pregnancy rates from all cryopreserved oocytes remain low. The inevitable use of the cryoprotectants (CPAs) during preservation affects the viability of the preserved oocytes and pregnancy rates either through CPA toxicity or osmotic injury. Current protocols attempt to reduce CPA toxicity by minimizing CPA concentrations, or by minimizing the volume changes via the step-wise addition of CPAs to the cells. Although the step-wise addition decreases osmotic shock to oocytes, it unfortunately increases toxic injuries due to the long exposure times to CPAs. To address limitations of current protocols and to rationally design protocols that minimize the exposure to CPAs, we developed a microfluidic device for the quantitative measurements of oocyte volume during various CPA loading protocols. We spatially secured a single oocyte on the microfluidic device, created precisely controlled continuous CPA profiles (step-wise, linear and complex) for the addition of CPAs to the oocyte and measured the oocyte volumetric response to each profile. With both linear and complex profiles, we were able to load 1.5 M propanediol to oocytes in less than 15 min and with a volumetric change of less than 10%. Thus, we believe this single oocyte analysis technology will eventually help future advances in assisted reproductive technologies and fertility preservation.     

Pyrazolyl methyls prescribe the electronic properties of iron(II) tetra(pyrazolyl)lutidine chloride complexes

A series of iron(II) chloride complexes of pentadentate ligands related to α,α,α',α'-tetra(pyrazolyl)-2,6-lutidine, pz(4)lut, has been prepared to evaluate whether pyrazolyl substitution has any systematic impact on the electronic properties of the complexes. For this purpose, the new tetrakis(3,4,5-trimethylpyrazolyl)lutidine ligand, pz**(4)lut, was prepared via a CoCl(2)-catalyzed rearrangement reaction. The equimolar combination of ligand and FeCl(2) in methanol gives the appropriate 1:1 complexes [FeCl(pz(R)(4)lut)]Cl that are each isolated in the solid state as a hygroscopic solvate. In solution, the iron(II) complexes have been fully characterized by several spectroscopic methods and cyclic voltammetry. In the solid state, the complexes have been characterized by X-ray diffraction, and, in some cases, by M?ssbauer spectroscopy. The M?ssbauer studies show that the complexes remain high spin to 4 K and exclude spin-state changes as the cause of the surprising solid-state thermochromic properties of the complexes. Non-intuitive results of spectroscopic and structural studies showed that methyl substitution at the 3- and 5- positions of the pyrazolyl rings reduces the ligand field strength through steric effects whereas methyl substitution at the 4-position of the pyrazolyl rings increases the ligand field strength through inductive effects.     

Synthesis of uronic-noeurostegine--a potent bacterial β-glucuronidase inhibitor

Inhibition of β-glucuronidases has recently been shown to be useful in alleviating drug toxicity for common colon cancer chemotherapeutic CPT-11 (also called Irinotecan). We have prepared a new compound of the nortropane-type, uronic-Noeurostegine, and demonstrated that this is a competitive and potent E. coli β-glucuronidase inhibitor, while inhibition of the mammalian β-glucuronidase from bovine liver was found to be less significant. Although not intended, two other compounds having N-ethyl and N-(4-hydroxybutyl) substituents were also prepared in this study due to the sluggish debenzylation in the final step. The N-substituents are believed to come from reaction with the solvents used being ethanol and THF, respectively. These compounds also inhibited the two β-glucuronidases albeit to a lesser extent compared to the parent compound. Noeurostegine and the three uronic-noeurostegines were additionally evaluated as inhibitors against a wide panel of glycosidases with the former showing potent inhibition of rat intestinal lactase and trehalase, whereas the latter was found to be inactive.     

Polycationic glycosides

Cationic lipids have long been known to serve as antibacterial and antifungal agents. Prior efforts with attachment of cationic lipids to carbohydrate-based surfaces have suggested the possibility that carbohydrate-attached cationic lipids might serve as antibacterial and antifungal pharmaceutical agents. Toward the understanding of this possibility, we have synthesized several series of cationic lipids attached to a variety of glycosides with the intent of generating antimicrobial agents that would meet the requirement for serving as a pharmaceutical agent, specifically that the agent be effective at a very low concentration as well as being biodegradable within the organism being treated. The initial results of our approach to this goal are presented.     

The mechanism of cleavage and isomerisation of RNA promoted by an efficient dinuclear Zn2+ complex

The cleavage and isomerisation of uridine 3'-alkylphosphates was studied in the presence of a dinuclear Zn(2+) complex, 3. The rate acceleration of the cleavage by 1 mM 3 is approximately 10(6)-fold under neutral conditions. Most remarkably, the complex also promotes the isomerisation of phosphodiester bonds, although the rate-enhancement is more modest: under neutral conditions complex 3 (1 mM) catalyses isomerisation by about 500-fold. The observation of this reaction shows that the reactions of these substrates catalysed by 3 proceed through a stepwise mechanism involving an intermediate phosphorane. A β(lg) value of -0.92 was determined for the 3-promoted cleavage reaction, and modest kinetic solvent deuterium isotope effects ranging from 1.5 to 2.8 were observed. Isomerisation was less sensitive to the nature of the esterifying group, with a β value of -0.5, and the kinetic solvent deuterium isotope effects were less than 1.5. Most of these characteristics of the 3-promoted cleavage are very similar to those for the cleavage of nucleoside 3'-phosphotriesters. These data are explained by a mechanism in which the complex primarily acts as an electrophilic catalyst neutralising the charge on the phosphate and stabilising an intermediate phosphorane, with general acid catalysis promoting the cleavage reaction. In contrast to the behaviour of triesters, isomerisation is significantly slower than cleavage; this suggests that the changes in geometry that occur during isomerisation lead to a much less stable complex between 3 and the phosphorane intermediate.     

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

The determination of total deoxyribonucleic acid (DNA) concentration is of great importance in many biological and bio-medical analyses. The quantification of DNA is traditionally performed by UV spectroscopy; however the results can be affected greatly by the sample matrix. The proposed method quantifies phosphorus in digested calf thymus DNA and human DNA by high performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS). The method presented showed excellent baseline separation between all four DNA mono-nucleotides and 5′UMP. The ability of LC-ICP-MS to provide an internal check that only DNA derived phosphorus was counted in the assay was demonstrated by establishing a mass balance between the total phosphorous signal from undigested DNA and that from the speciated DNA. Column recoveries ranging from 95% to 99% for phosphorus resulted in a mass balance of 95% ± 0.5% for standard nucleotides, determined by LC-ICP-MS, compared to total DNA determined by flow injection coupled to ICP-MS (FI-ICP-MS). The method for quantification was validated by analysis of NIST SRM 2,372; a total speciated DNA recovery of 52.1 ng/μL, compared with an expected value of 53.6 ng/μL, was determined by external calibration. From repeat measurements, a mass balance of 97% ± 0.5% for NIST DNA was achieved. The method limits of detection for individual nucleotides were determined between 0.8 and 1.7 μg L⁻¹ (³¹P) for individual nucleotides by LC-ICP-MS, and 360 ng L⁻¹ for 5′AMP by direct nebulisation.     

Microclimate monitoring in the Carcer Tullianum: temporal and spatial correlation and gradients evidenced by multivariate analysis; first campaign

Too often microclimate studies in the field of cultural heritage are published without any or scarce information on sampling design, sensors (type, number, position) and instrument validation. Lacking of this fundamental information does not allow an open discussion in the scientific community. This work aims to be an invitation for a different approach.Three main parameters (temperature, humidity, luminance) were monitored in a selected part of a complex construction by an inexpensive self-assembled system along some horizontal and vertical vectors. All data was then processed and analyse by chemometric methods. Some measurements of oxygen, carbon monoxide and dioxide and pressure were also performed.Correlation of some indoor and outdoor data was shown for temperature and humidity. In case of outdoor changes the indoor environment reacted with a certain delay which is position-dependent and more evident for humidity data. The two observed rooms (Carcer and Tullianum) behave differently and the hypogean one is less influenced by the outdoor environment. Instrument validation before and after the campaign, allows to consider detected variations as significant.The fundamental importance of Sampling Design and of instrument validation before and after the monitoring campaign was enhanced. The choice of two main and two minor vectors allowed detection of different behaviour for the two rooms, also permitting to detect for both rooms a trend towards a spontaneous microclimate necessary for a conservation project. In the next campaign we will focus on the choice of the best sampling frequency to use more sophisticated statistical methods.