Towards identifying the mode of action of drugs using live-cell FTIR spectroscopy.

Fourier transform infrared spectroscopy (FTIR) has been shown to be a promising tool for identifying the mode of action of drugs. However, most previous studies have focused on the analysis of fixed or dried cells. The measurement of living cells has the advantage of obtaining time series data, and the in situ approach eliminates the need for fixing or drying the cells. In this study, the potential of live-cell FTIR method for the identification of the mode of action of drugs was demonstrated. Four different drugs were tested, with two of the drugs having the same mode of action (tamoxifen and toremifene) and the other two having different modes of action (imatinib and doxorubicin). Live cells were treated in the four drugs at and below the IC50 level (i.e. the concentration of drug required to inhibit the growth of cells by 50%), and the changes to their spectra after the addition of drugs were monitored over a 24-hour period. Principal component analysis (PCA) of the spectral data shows that drugs with different modes of action are well-separated, while the drugs with the same mode of action are grouped together. The results also show that at IC50, the separation appears to be the clearest at 2 hours for imatinib and tamoxifen/toremifene and 6 hours for doxorubicin. However, at 50% of the IC50 drug concentration, the separation appears to be the best at longer incubation time, i.e. 24 hours, for all four drugs. In conclusion, live-cell FTIR has shown to be able to distinguish and group spectral signatures of cells treated with drugs of known modes of action after a relatively short time of exposure. Further collection of live-cell data would enable an algorithm to be developed for the prediction of the modes of action of novel drugs, which can help in the preclinical drug screening process.

Questionnaire survey on medical futility and termination of resuscitation in cardiac arrest patients among emergency physicians in Hong Kong.

INTRODUCTION: The perceptions of medical futility and decisions about termination of resuscitation (TOR) for out-of-hospital cardiac arrest (OHCA) are highly heterogeneous and dependent on the practice of the attending emergency physicians. The objective of this study was to report and investigate the knowledge, attitudes, and practices regarding medical futility and TOR during management of OHCA in Hong Kong. METHODS: A cross-sectional survey was conducted among emergency medicine physicians in Hong Kong. The questionnaire assessed participants' background, knowledge, attitudes, and behaviours concerning medical futility and TOR in management of OHCA. Composite scores were calculated to reflect knowledge, attitudes, and practices of OHCA treatment. Subgroup analysis and multiple regression analysis were used to explore the relationship between participants' background, knowledge, attitudes, and behaviours. RESULTS: The response rate to this survey was 57% (140/247). Independent predictors of less aggressive resuscitation in OHCA patients included status as a Fellow of the Hong Kong College of Emergency Medicine (ß= -0.314, P=0.028) and being an Advanced Cardiac Life Support instructor (ß= -0.217, P=0.032). There was no difference in aggressiveness of resuscitation in terms of years of clinical experience (ß=0.015, P=0.921), knowledge of TOR (ß=0.057, P=0.509), or attitudes about TOR (ß= -0.103, P=0.214). The correlation between knowledge and attitudes was low (Spearman's coefficient=0.02, P=0.795). CONCLUSION: Clinical practice and behaviour of TOR was not demonstrated to have associations with knowledge or attitude. Status as a Fellow of the Hong Kong College of Emergency Medicine or Advanced Cardiac Life Support instructor were the only two parameters identified that had significant relationships with earlier TOR in medically futile patients with OHCA.

Effect of immunosuppressive therapies on survival of systemic lupus erythematosus: a propensity score analysis of a longitudinal cohort.

Objectives The aim of this study was to study the relationship between immunosuppressive drug treatment and survival in patients with systemic lupus erythematosus (SLE). Methods Patients who fulfilled four or more American College of Rheumatology criteria for SLE were followed longitudinally. Clinical characteristics, use of immunosuppressive agents and mortality were reviewed. Cox regression was used to study the relationship between immunosuppressive treatment and survival, adjusted for age, sex, vascular risk factors, organ damage, the anti-phospholipid antibodies and a propensity score for the indication of individual immunosuppressive agent derived from separate regression models. Results A total of 803 SLE patients were studied (92% women; age of SLE onset 33.2±14 years; follow-up time 10.8±7.7 years). The frequencies of ever use of immunosuppressive agents were: high-dose prednisolone (≥0.6 mg/kg/day for ≥4 weeks) (85%), azathioprine (63%), cyclophosphamide (25%), mycophenolate mofetil (27%), the calcineurin inhibitors (23%) and hydroxychloroquine (69%). Ninety-seven patients (12%) died and 56 (7%) patients were lost to follow-up. The causes of death were infection (44%), cerebrovascular events (12%), cardiovascular events (10%) and malignancy (8.2%). Cox regression revealed that the ever use of high-dose prednisolone, mycophenolate mofetil, calcineurin inhibitors or cyclophosphamide was not significantly associated with improved survival. However, the ever use of hydroxychloroquine (hazard ratio 0.59 (0.37-0.93); P=0.02) and azathioprine (hazard ratio 0.46 (0.28-0.75); P=0.002) was significantly associated with reduced mortality (41% and 54%, respectively) after adjustment for the propensity score and other confounding factors. A similar beneficial effect of hydroxychloroquine and azathioprine on survival was also observed in patients with lupus nephritis. Conclusions In this longitudinal cohort of Chinese SLE patients, the ever use of hydroxychloroquine and azathioprine was significantly associated with a probability of better survival. Treatment with high-dose prednisolone, cyclophosphamide, mycophenolate mofetil or the calcineurin inhibitors was not associated with long-term survival benefit.

Subcellular mapping of living cells via synchrotron microFTIR and ZnS hemispheres.

FTIR imaging is a label-free, non-destructive method valuably exploited in the study of the biological process in living cells. However, the long wavelength/low spatial resolution and the strong absorbance of water are still key constrains in the application of IR microscopy ex vivo. In this work, a new retrofit approach based on the use of ZnS hemispheres is introduced to significantly improve the spatial resolution on live cell FTIR imaging. By means of two high refractive index domes sandwiching the sample, a lateral resolution close to 2.2 µm at 6 µm wavelength has been achieved, i.e. below the theoretical diffraction limit in air and more than twice the improvement (to ~λ/2.7) from our previous attempt using CaF2 lenses. The ZnS domes also allowed an extended spectral range to 950 cm-1, in contrast to the cut-off at 1050 cm-1 using CaF2. In combination with synchrotron radiation source, microFTIR provides an improved signal-to-noise ratio through the circa 12 µm thin layer of medium, thus allowing detailed distribution of lipids, protein and nucleic acid in the surround of the nucleus of single living cells. Endoplasmic reticula were clearly shown based on the lipid ν(CH) and ν(C=O) bands, while the DNA was imaged based on the ν(PO2-) band highlighting the nucleus region. This work has also included a demonstration of drug (doxorubicin) in cell measurement to highlight the potential of this approach. Graphical abstract.

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) imaging of tissues and live cells.

FTIR spectroscopic imaging is a label-free, non-destructive and chemically specific technique that can be utilised to study a wide range of biomedical applications such as imaging of biopsy tissues, fixed cells and live cells, including cancer cells. In particular, the use of FTIR imaging in attenuated total reflection (ATR) mode has attracted much attention because of the small, but well controlled, depth of penetration and corresponding path length of infrared light into the sample. This has enabled the study of samples containing large amounts of water, as well as achieving an increased spatial resolution provided by the high refractive index of the micro-ATR element. This review is focused on discussing the recent developments in FTIR spectroscopic imaging, particularly in ATR sampling mode, and its applications in the biomedical science field as well as discussing the future opportunities possible as the imaging technology continues to advance.

A central neural pathway controlling odor tracking in Drosophila.

Chemotaxis is important for the survival of most animals. How the brain translates sensory input into motor output beyond higher olfactory processing centers is largely unknown. We describe a group of excitatory neurons, termed Odd neurons, which are important for Drosophila larval chemotaxis. Odd neurons receive synaptic input from projection neurons in the calyx of the mushroom body and project axons to the central brain. Functional imaging shows that some of the Odd neurons respond to odor. Larvae in which Odd neurons are silenced are less efficient at odor tracking than controls and sample the odor space more frequently. Larvae in which the excitability of Odd neurons is increased are better at odor intensity discrimination and odor tracking. Thus, the Odd neurons represent a distinct pathway that regulates the sensitivity of the olfactory system to odor concentrations, demonstrating that efficient chemotaxis depends on processing of odor strength downstream of higher olfactory centers.

In situ Fourier transform infrared analysis of live cells' response to doxorubicin.

The study of the response of cancer cells to chemotherapy drugs is of high importance due to the specificity of some drugs to certain types of cancer and the resistance of some specific cancer types to chemotherapy drugs. Our aim was to develop and apply the label-free and non-destructive Fourier transform infrared (FTIR) method to determine the sensitivity of three different cancer cell-lines to a common anti-cancer drug doxorubicin at different concentrations and to demonstrate that information about the mechanism of resistance to the chemotherapy drug can be extracted from spectral data. HeLa, PC3, and Caco-2 cells were seeded and grown on an attenuated total reflection (ATR) crystal, doxorubicin was applied at the clinically significant concentration of 0.1-20 µM, and spectra of the cells were collected hourly over 20 h. Analysis of the amide bands was correlated with cell viability, which had been cross validated with MTT assays, allowing to determine that the three cell lines had significantly different resistance to doxorubicin. The difference spectra and principal component analysis (PCA) highlighted the subtle chemical changes in the living cells under treatment. Spectral regions assigned to nucleic acids (mainly 1085 cm(-1)) and carbohydrates (mainly 1024 cm(-1)) showed changes that could be related to the mode of action of the drug and the mechanism of resistance of the cell lines to doxorubicin. This is a cost-effective method that does not require bioassay reagents but allows label-free, non-destructive and in situ analysis of chemical changes in live cells, using standard FTIR equipment adapted to ATR measurements.

A prospective cross-sectional study of anxiety and depression in patients with psoriasis in Singapore.

BACKGROUND: Psoriasis has a negative psychological impact on patients, and may have repercussions on treatment outcomes. Despite this, the degree to which psoriatic patients suffer from psychiatric disorders has not received much attention in Singapore. OBJECTIVE: This prospective cross-sectional study was conducted to determine the frequency of anxiety and depression in a cohort of Singaporean patients with psoriasis, and explore its relationship with regards to physical disease severity and subjective quality of life. METHODS: 100 patients aged 21-60 years old who visited the National Skin Centre, Singapore from 2008 to 2009 were enrolled into the study. Anxiety and depression were quantified using the Hospital Anxiety and Depression Scale (HADS). Disease severity was quantified with the Psoriasis Area Severity Index (PASI) and quality of life measured with the Short Form (36) Health Survey (SF-36). RESULTS: Using the HADS, the mean score for anxiety was 6.9 and that for depression was 4.7. An anxiety disorder was suggested in 17%, while a depressive disorder was suggested in 15% of the study population. All eight domains of the SF-36 were significantly correlated with both anxiety and depression scores. Patients with moderate or severe psoriasis (on PASI) had worse depression scores than those with mild psoriasis. No association was found between anxiety scores and PASI. Neither was any significant correlation seen between anxiety and depression scores vs. patients' age, monthly income and duration of psoriasis. CONCLUSION: This study demonstrates the strong psychiatric morbidity in patients with psoriasis, for which further psychiatric evaluation should be considered.

Effect of golimumab and pamidronate on clinical efficacy and MRI inflammation in axial spondyloarthritis: a 48-week open randomized trial.

OBJECTIVES: To compare the effect of golimumab (GLM) and pamidronate (PAM) on clinical efficacy and magnetic resonance imaging (MRI) inflammation in axial spondyloarthritis (aSpA). METHOD: Patients who fulfilled the Assessment of SpondyloArthritis Society (ASAS) criteria for aSpA and had active disease [Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score ≥ 4] were randomized in a 2:1 ratio to receive either GLM (50 mg) or PAM (60 mg) 4 weekly for 48 weeks. Clinical efficacy was assessed at intervals. Inflammation of the spine and sacroiliac joints (SIJs) on MRI was graded by the Spondyloarthritis Research Consortium of Canada (SPARCC) scoring system. RESULTS: Twenty patients were assigned to GLM and 10 to PAM (83% men; age 33.4 ± 10.9 years; disease duration 4.4 ± 3.4 years). The baseline characteristics of the two groups were similar. At week 48, the proportions of patients who achieved an ASAS20 response were not significantly different between the GLM and PAM groups (65% vs. 56%; p = 0.69). Although there were no differences in BASDAI, spinal pain, and Medical Outcomes Study 36-item Short Form Health Survey (SF-36) scores between the two groups at week 48, the Ankylosing Spondylitis Disease Activity Score (ASDAS), Bath AS Functional Index (BASFI), C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) levels were significantly lower in GLM-treated patients. The SPARCC scores of the spine and SIJs decreased significantly in GLM- but not in PAM-treated patients. The differences in SPARCC scores between the two groups at week 48 were statistically significant. The frequency of adverse events (AEs) was similar in both arms. CONCLUSIONS: In patients with aSpA, the clinical response rate and improvement in pain and quality of life (QoL) were similar between GLM and PAM groups after 48 weeks. However, significant reduction in inflammatory markers and MRI inflammation was only observed with GLM treatment.

Fetal brain injury in survivors of twin pregnancies complicated by demise of one twin as assessed by in utero MR imaging.

OBJECTIVE: The aim of this study was to quantify the risk of brain abnormalities in single-twin demise of monochorionic pregnancies and to describe the type of brain injuries using ultrasound and in utero magnetic resonance (iuMR) imaging. METHODS: Monochorionic twin pregnancies complicated by the demise of one twin referred between 2004 and 2013 were reviewed. Ultrasound was performed in a tertiary centre prior to iuMR. The cases were subdivided into those associated with co-twin loss following fetoscopic laser ablative treatment for twin-twin transfusion syndrome (TTTS) and those associated with spontaneous fetal demise. RESULTS: Sixty-eight cases were identified, 27/68 following treatment for TTTS and 41/68 with spontaneous fetal demise. Nine (13.2%) had brain abnormalities on iuMR, and the rate of brain abnormalities was similar in the two groups. Expert ultrasound and iuMR findings agreed in three out of nine of those cases, and in six out of nine cases, ultrasound underestimated or missed the pathology. CONCLUSION: Monochorionic twin pregnancies with single fetal demise are complex pregnancies with increased risk of acquired brain pathology, although the rate of brain abnormalities in our study is lower than that of other publications. iuMR in such complicated pregnancies is a useful adjuvant imaging technique that appears to detect brain pathologies better than prenatal ultrasonography.

Label-free in situ quantification of drug in living cells at micromolar levels using infrared spectroscopy.

Quantifying the rate and the amount of drug entering live cells is an essential part of the medicine development process. Infrared spectroscopy is a label-free, chemically selective tool for analyzing the composition of live cells in culture that has the potential to quantify, in situ, the amount of drug entering living cells in a nondestructive manner, although its sensitivity is currently limited. This paper is the first to demonstrate in situ quantification of the cancer drug, fluorouracil, in live cells at a therapeutically relevant concentration using Fourier transform infrared spectroscopy. To achieve the required improvement in detection and quantitation limits of the IR measurement, two strategies were exploited. First, a sampling method called multibounce attenuated total reflection was used to optimize the signal while second, a long pass filter in combination with a mercury cadmium telluride detector was used to reduce the instrument noise. Using these novel adaptations, it was possible to quantify 20 µM of fluorouracil in cell culture medium using a standard FTIR instrument, while it was possible to quantify and measure the flux of fluorouracil in situ in living cells treated with an 80 µM drug.

Manufacture and Initial Characterisation of RAPIDTM Biodynamic Haematogel, an Autologous Platelet and Leukocyte-Rich Plasma Gel for Diabetic Foot Ulcers.

This observational study reports the process for the manufacture of RAPIDTM Biodynamic Haematogel and explores the properties of the platelet and leukocyte-rich plasma gels formed. Gels were manufactured from 60 mL of human blood using the protocol of Biotherapy Services. Platelet and leukocyte content, time-to-gel, gel weight and the temporal profile of liquid exudation from the gels were measured, along with the content of growth factors VEGF and PDGF in the releasate. The effect of the releasate on human keratinocyte (HaCat) cell proliferation was also determined. The platelet and leukocyte concentrations in donor blood were 1.60-8.10 × 108 and 1.00 × 106-2.00 × 107 cells/mL, which were concentrated 2.67- and 1.12-fold, respectively, during processing. Structurally weak gels were formed which exuded a clear liquid releasate (77.4% w/w of gel weight over 60 min) that contained 278 pg/mL VEGF and 1319 pg/mL PDGF. The releasate produced concentration-dependent proliferation of HaCat cells: 5-15% releasate produced a 2.7-8.9-fold increase in growth over 48 h. In conclusion, we have described the point-of-care manufacturing protocol and characterised the gel properties of RAPIDTM Biodynamic Haematogel. This is an essential first step towards identifying, understanding and controlling critical processing parameters that impact on this medicinal product's quality.

Expression of anti-inflammatory macrophage genes within skeletal muscle correlates with insulin sensitivity in human obesity and type 2 diabetes.

AIMS/HYPOTHESIS: Low-grade systemic inflammation and adipose tissue inflammatory macrophages are frequently detected in patients with obesity and type 2 diabetes. Whether inflammatory macrophages also increase in skeletal muscle of individuals with metabolic disorders remains controversial. Here, we assess whether macrophage polarisation markers in skeletal muscle of humans correlate with insulin sensitivity in obesity and type 2 diabetes. METHODS: Skeletal muscle biopsies were obtained from individuals of normal weight and with normal glucose tolerance (NGT), and overweight/obese individuals with or without type 2 diabetes. Insulin sensitivity was determined by euglycaemic-hyperinsulinaemic clamps. Expression of macrophage genes was analysed by quantitative RT-PCR. RESULTS: Gene expression of the inflammatory macrophage phenotype marker cluster of differentiation (CD)11c was higher in muscle of type 2 diabetes patients (p = 0.0069), and correlated with HbA1c (p = 0.0139, ρ = 0.48) and fasting plasma glucose (p = 0.0284, ρ = 0.43), but not after correction for age. Expression of TGFB1, encoding the anti-inflammatory marker TGF-ß1, correlated inversely with HbA1c (p = 0.0095, ρ = -0.50; p = 0.0484, ρ = -0.50) and fasting plasma glucose (p = 0.0471, ρ = -0.39; p = 0.0374, ρ = -0.52) in two cohorts, as did HbA1c with gene expression of macrophage galactose-binding lectin (MGL) (p = 0.0425, ρ = -0.51). TGFB1 expression was higher in NGT individuals than in individuals with type 2 diabetes (p = 0.0303), and correlated with low fasting plasma insulin (p = 0.0310, ρ = -0.42). In exercised overweight/obese individuals, expression of genes for three anti-inflammatory macrophage markers, MGL (p = 0.0031, ρ = 0.71), CD163 (p = 0.0268, ρ = 0.57) and mannose receptor (p = 0.0125, ρ = 0.63), correlated with high glucose-disposal rate. CONCLUSIONS/INTERPRETATION: Muscle expression of macrophage genes reveals a link between inflammatory macrophage markers, age and high glycaemia, whereas anti-inflammatory markers correlate with low glycaemia and high glucose-disposal rate.

Correcting the effect of refraction and dispersion of light in FT-IR spectroscopic imaging in transmission through thick infrared windows.

Transmission mode is one of the most common sampling methods for FT-IR spectroscopic imaging because the spectra obtained generally have a reasonable signal-to-noise ratio. However, dispersion and refraction of infrared light occurs when samples are sandwiched between infrared windows or placed underneath a layer of liquid. Dispersion and refraction cause infrared light to focus with different focal lengths depending on the wavelength (wavenumber) of the light. As a result, images obtained are in focus only at a particular wavenumber while they are defocused at other wavenumber values. In this work, a solution to correct this spread of focus by means of adding a lens on top of the infrared transparent window, such that a pseudo hemisphere is formed, has been investigated. Through this lens (or pseudo hemisphere), refraction of light is removed and the light across the spectral range has the same focal depth. Furthermore, the lens acts as a solid immersion objective and an increase of both magnification and spatial resolution (by 1.4 times) is demonstrated. The spatial resolution was investigated using an USAF resolution target, showing that the Rayleigh criterion can be achieved, as well as a sample with a sharp polymer interface to indicate the spatial resolution that can be expected in real samples. The reported approach was used to obtain chemical images of cross sections of cancer tissue and hair samples sandwiched between infrared windows showing the versatility and applicability of the method. In addition to the improved spatial resolution, the results reported herein also demonstrate that the lens can reduce the effect of scattering near the edges of tissue samples. The advantages of the presented approach, obtaining FT-IR spectroscopic images in transmission mode with the same focus across all wavenumber values and simultaneous improvement in spatial resolution, will have wide implications ranging from studies of live cells to sorption of drugs into tissues.

Development of systemic lupus erythematosus in a male-to-female transsexual: the role of sex hormones revisited.

Systemic lupus erythematosus (SLE) predominantly affects women of childbearing age. The infrequency of SLE in men and disease onset in prepubertal or postmenopausal women suggests a role of estrogen in the predisposition to the disease. Patients with hypergonadotrophic hypogonadism are prone to the development of SLE, and the use of exogenous estrogens in women increases the relative risk of SLE onset and disease flares. These observations provide indirect evidence for an opposite role of estrogens and androgens in the pathogenesis of SLE. We report on a male-to-female transsexual who developed SLE 20 years after sex-reassignment surgery and prolonged estrogen therapy. The role of sex hormones in SLE is revisited.

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

ATR-FTIR spectroscopic imaging: recent advances and applications to biological systems.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging is a highly versatile, label free and non-destructive chemical imaging method which can be applied to study a wide range of samples and systems. This review summarises some of the recent advances and applications of this imaging method in the area of biomedical studies, including examples of section of aorta, skin tissue and live cells. Two of the major advantages of measuring in ATR mode are the opportunity to measure samples that absorb strongly in the IR spectrum, such as aqueous systems, without significant sample preparation and the ability to increase the spatial resolution of the measured image. The implications of these advantages as well as some limitations of this imaging approach are discussed and a brief outlook at some of the possible future developments in this area is provided.