Priorities for research promoting mental health in the south and east of Asia.

Progress in promoting mental health, preventing mental illness, and improving care for people affected by mental illness is unlikely to occur if efforts remain separated from existing public health programs and the principles of public health action. Experts met recently to discuss integrating public health and mental health strategies in the south and east of Asia, especially in low- and middle-income countries. Areas of research identified as high priority were: 1) integrating mental health into perinatal care; 2) providing culturally-adjusted support for carers of people with mental and physical disorders; 3) using digital health technologies for mental health care in areas with limited resources and 4) building local research capacity. Selection of these areas was informed by their relative novelty in the region, ease of implementation, likely widespread benefit, and potential low costs. In this article, we summarise available evidence, highlight gaps and call for collaborations with research centres, leaders and persons with lived experience within and beyond the region.

Anxiolytic potential of resveratrol and rufinamide combination by modulating GABA-ergic transmission: insights from experiments, molecular docking and dynamics simulations.

Resveratrol is a polyphenolic phytocompound known to possess anxiolytic-like effects but its impact on central gammaaminobutyric acid (GABA) modulation has never been explored. The purpose of this study was to analyze the anxiolytic-like effects of resveratrol alone and in combination with rufinamide, an antiepileptic drug which has never been studied for its anxiolytic potential. The BALB/c mice were tested in a battery of behavior testing after administration of resveratrol (50 mg/kg) and rufinamide (50 mg/kg) alone and in combination. Moreover, molecular docking studies were also carried out to understand the interaction of resveratrol and rufinamide with GABA aminotransferase, GABA receptor and GABA-A transporter type 1. Resveratrol alone exerted notable anxiolytic-like effects and improved outcomes in few experiments but rufinamide alone did not yield any beneficial outcomes. However, the animal co-administered with resveratrol and rufinamide behaved exceptionally well (p<0.05) and preferred open, illuminated and exposed areas of open field, light/dark and elevated plus maze. Further, these animals showed reduced anxiety towards anxiogenic stimuli i.e. holes and marbles in hole board and marble bury tests, respectively. Resveratrol and rufinamide showed moderate to strong binding affinities with GABA proteins, indicating the potential to treat anxiety-like neurological disorders. Moreover, resveratrol and rufinamide were analyzed using molecular docking to determine their interaction with GABA receptors, transporters, and transaminase. The results suggest that their anxiolytic-like effects may be due to inhibiting GABA reuptake transporter 1 protein, leading to increased synaptic levels of GABA neurotransmitter, as seen in stable molecular dynamics results with the 7SK2 GABA transporter protein.

Neuropharmacological investigation, ultra-high performance liquid chromatography analysis, and in silico studies of Phyla nodiflora.

The increasing burden of neurological disorders is becoming a worldwide health challenge and researchers are continuously struggling to cure them by utilizing the miraculous medicinal properties of plants. The crude methanolic extract of whole herb of Phyla nodiflora (Pn.Cr) was subjected to phytochemical, antioxidant and neuropharmacological assessment. The Pn.Cr was initially exposed to the in vitro examination for phytocomposition through ultra-high performance liquid chromatography (UHPLC). The Sprague Dawley rats were chronically administered with various doses (100, 200 and 300 mg/kg) of Pn.Cr for one month with subsequent exposure to neurobehavioral and biochemical experimentation. The Pn.Cr exhibited a dose-dependent anxiolytic effect (P < 0.05 in comparison to control) as rats preferred central, illuminated and open arm zones in open field (OFT), light/dark (L/D) and elevated plus maze (EPM) tests. Likewise, scopolamine-induced amnesia was noticeably reversed with P < 0.05 by Pn.Cr as animals showed improved spontaneous alternation, discrimination index and shorter escape latencies in Y-maze, novel object recognition (NOR) and Morris water maze (MWM) tests. Subsequently, in vivo enzymatic assays depicted the reduced acetylcholinesterase and malondialdehyde levels. The levels of oxidative stress combating enzymes (glutathione peroxidase and superoxide dismutase) were increased in a dose-dependent style. The UHPLC detected 22 phytocompounds were further investigated in silico studied to predict the interaction of blood-brain barrier (BBB) crossing phytocompounds with human acetylcholinesterase. The four BBB crossing phytocompounds belonging to flavonoids, chalcones and alkaloids showed possible interaction with the target enzyme. We found that the phytocompounds owned by Pn.Cr might be playing multiple roles in modulation of different pathways to hinder the pathophysiology of neurological disorders including anxiety and Alzheimer's disease.

Time course evaluation of lacosamide alone and in polypharmacy on behavioral manifestations and oxidative stress in lithium-pilocarpine-induced model.

The lithium-pilocarpine model in rats is commonly used to study the characteristic events of acute status epilepticus (SE), epileptogenesis and temporal lobe epilepsy (TLE). Here we investigated the impact of lacosamide alone and in combination with other drugs (pregabalin, piracetam and scopolamine) on spontaneous recurrent seizures (SRSs) and behavioral parameters during the time frame of 6 weeks after SE. In addition, the level of oxidative stress in the hippocampus was accessed by real-time microdialysis study (8-isoprostanes) and antioxidants enzymes in the homogenate. Results revealed severe behavioral deficits with the control epileptic group and animals displayed hyperexcitability, aggression apprehension and memory insufficiency. Pharmacological manipulation for 6 weeks with lacosamide (L) - 80 mg/kg; in polypharmacy with pregabalin (L/P) - 50/50 mg/kg and piracetam (L/Pi) - 50/140 mg/kg significantly (P < 0.05) ameliorated the anxiety-related behavior (open filed, elevated plus maze, light/dark tests), depression (forced swim test) and improved spatial/reference memory (Morris water maze). There were low incidences of seizures in L, L/P and L/Pi groups revealing disease-modifying effects of employed drugs. Furthermore, the chronic use of scopolamine (L/P/S; 50/50/2 mg/kg) as polypharmacy with the concept of antagonizing the cholinergic inputs in the epileptogenic phase aberrated the behavioral situation further worse. Treatments with L/P and L/Pi significantly attenuated (P < 0.05) the oxidative stress by reducing 8-isoprostanes and malondialdehyde (MDA) levels. Furthermore, superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels in the L/P group were significantly (P < 0.05) improved. Overall, our findings support the use of a combination of drugs (L/P and L/Pi) in lithium-pilocarpine model which remarkably ameliorated SRSs, reduced anxiety-related behaviors, retention of spatial/reference memory and lowered oxidative stress in a time-course evaluation 6 weeks post- SE insult.

Fingerprint-to-CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope.

Stimulated Raman scattering (SRS) microscopy is a label-free method generating images based on chemical contrast within samples, and has already shown its great potential for high-sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio-samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub-cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto-optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon-Hydrogen (CH)-stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm-1 , is demonstrated.

Manual preoperative tracheal retraction exercise decreases the occurrence of postoperative oropharyngeal dysphagia after anterior cervical discectomy and fusion.

OBJECTIVE: Preoperative tracheal retraction exercise (TRE) to minimize the occurrence of postoperative oropharyngeal dysphagia after anterior cervical spine surgery. METHODS: A total of 220 patients admitted for elective anterior cervical spine surgery from January 2013 to December 2014 were retrospectively reviewed. The patients were allocated into two groups: TRE group and control group (without TRE). Modified dysphagia scoring system (MDSS) was used for evaluating the presence and severity of dysphagia symptoms at 1 week and 1, 3, and 6 months after surgery. Demographics such as age, gender, smoking, type of procedure, number of levels operated, duration of surgery, intraoperative blood loss, and instrumentation were analyzed. The clinical outcomes in both groups were compared with Neck Disability Index (NDI), Visual Analogue Scale (VAS) for arm and neck pain, and Odom's criteria for global outcome. RESULTS: In the first week postoperatively, 86 patients (39.1%) developed dysphagia, which decreased to 72 (32.7%), 5 (2.3%), and 4 (1.8%) after 1, 3, and 6 months, respectively. The patients who received the TRE prior to surgery had significantly better MDSS scores ( p = 0.032 for second-level, 0.022 for third-level, and 0.009 for fourth-level fusions) than control group patients who did not receive TRE at the first week of surgery. At the 1-month follow-up, the followed-up patients for second- to fourth-level fusions in the TRE group had improved MDSS scores than those in the control group ( p = 0.041 for second-level, 0.025 for third-level, and 0.0011 for fourth-level fusions). MDSS scores showed no significant difference between both the groups at 1 and 3 months postoperatively for single level anterior cervical fusion. NDI and VAS scores didn't yield any significant difference. Global outcome by Odom's criteria was 88.6%. CONCLUSION: Preoperative TRE can significantly reduce the occurrence of postoperative dysphagia after ACDF surgery. During follow-up, the incidence of postoperative dysphagia was significantly lower and had resolved at 3 months in all patients.

Curricular integration of social medicine: a prospective for medical educators.

In the United States, the health of a community falls on a continuum ranging from healthy to unhealthy and fluctuates based on several variables. Research policy and public health practice literature report substantial disparities in life expectancy, morbidity, risk factors, and quality of life, as well as persistence of these disparities among segments of the population. One such way to close this gap is to streamline medical education to better prepare our future physicians for our patients in underserved communities. Medical schools have the potential to close the gap when training future physicians by providing them with the principles of social medicine that can contribute to the reduction of health disparities. Curriculum reform and systematic formative assessment and evaluative measures can be developed to match social medicine and health disparities curricula for individual medical schools, thus assuring that future physicians are being properly prepared for residency and the workforce to decrease health inequities in the United States. We propose that curriculum reform includes an ongoing social medicine component for medical students. Continued exposure, practice, and education related to social medicine across medical school will enhance the awareness and knowledge for our students. This will result in better preparation for the zero mile stone residency set forth by the Accreditation Council of Graduate Medical Education and will eventually lead to the outcome of higher quality physicians in the United States to treat diverse populations.

Status of neurology medical school education: results of 2005 and 2012 clerkship director survey.

OBJECTIVE: To survey all US medical school clerkship directors (CDs) in neurology and to compare results from a similar survey in 2005. METHODS: A survey was developed by a work group of the American Academy of Neurology Undergraduate Education Subcommittee, and sent to all neurology CDs listed in the American Academy of Neurology database. Comparisons were made to a similar 2005 survey. RESULTS: Survey response rate was 73%. Neurology was required in 93% of responding schools. Duration of clerkships was 4 weeks in 74% and 3 weeks in 11%. Clerkships were taken in the third year in 56%, third or fourth year in 19%, and fourth year in 12%. Clerkship duration in 2012 was slightly shorter than in 2005 (fewer clerkships of ≥4 weeks, p = 0.125), but more clerkships have moved into the third year (fewer neurology clerkships during the fourth year, p = 0.051). Simulation training in lumbar punctures was available at 44% of schools, but only 2% of students attempted lumbar punctures on patients. CDs averaged 20% protected time, but reported that they needed at least 32%. Secretarial full-time equivalent was 0.50 or less in 71% of clerkships. Eighty-five percent of CDs were "very satisfied" or "somewhat satisfied," but more than half experienced "burnout" and 35% had considered relinquishing their role. CONCLUSION: Trends in neurology undergraduate education since 2005 include shorter clerkships, migration into the third year, and increasing use of technology. CDs are generally satisfied, but report stressors, including inadequate protected time and departmental support.

Surface-enhanced Raman spectroscopy of the endothelial cell membrane.

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.

Surface presentation of functional peptides in solution determines cell internalization efficiency of TAT conjugated nanoparticles.

Functionalizing nanoparticles with cell-penetrating peptides is a popular choice for cellular delivery. We investigated the effects of TAT peptide concentration and arrangement in solution on functionalized nanoparticles' efficacy for membrane permeation. We found that cell internalization correlates with the positive charge distribution achieved prior to nanoparticle encountering interactions with membrane. We identified a combination of solution based properties required to maximize the internalization efficacy of TAT-functionalized nanoparticles.

Determination using synchrotron radiation-based Fourier transform infrared microspectroscopy of putative stem cells in human adenocarcinoma of the intestine: corresponding benign tissue as a template.

The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 µm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as "distinct base region" spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations could point to a cancer SC phenotype; an approach capable of identifying their in situ location has enormous therapeutic applications.

A competency-based longitudinal core curriculum in medical neuroscience.

Current medical educational theory encourages the development of competency-based curricula. The Accreditation Council for Graduate Medical Education's 6 core competencies for resident education (medical knowledge, patient care, professionalism, interpersonal and communication skills, practice-based learning, and systems-based practice) have been embraced by medical schools as the building blocks necessary for becoming a competent licensed physician. Many medical schools are therefore changing their educational approach to an integrated model in which students demonstrate incremental acquisition and mastery of all competencies as they progress through medical school. Challenges to medical schools include integration of preclinical and clinical studies as well as development of learning objectives and assessment measures for each competency. The Undergraduate Education Subcommittee (UES) of the American Academy of Neurology (AAN) assembled a group of neuroscience educators to outline a longitudinal competency-based curriculum in medical neuroscience encompassing both preclinical and clinical coursework. In development of this curriculum, the committee reviewed United States Medical Licensing Examination content outlines, Liaison Committee on Medical Education requirements, prior AAN-mandated core curricula for basic neuroscience and clinical neurology, and survey responses from educators in US medical schools. The newly recommended curriculum provides an outline of learning objectives for each of the 6 competencies, listing each learning objective in active terms. Documentation of experiences is emphasized, and assessment measures are suggested to demonstrate adequate achievement in each competency. These guidelines, widely vetted and approved by the UES membership, aspire to be both useful as a stand-alone curriculum and also provide a framework for neuroscience educators who wish to develop a more detailed focus in certain areas of study.

Gold nanoparticles explore cells: cellular uptake and their use as intracellular probes.

Understanding uptake of nanomaterials by cells and their use for intracellular sensing is important for studying their interaction and toxicology as well as for obtaining new biological insight. Here, we investigate cellular uptake and intracellular dynamics of gold nanoparticles and demonstrate their use in reporting chemical information from the endocytotic pathway and cytoplasm. The intracellular gold nanoparticles serve as probes for surface-enhanced Raman spectroscopy (SERS) allowing for biochemical characterisation of their local environment. In particular, in this work we compare intracellular SERS using non-functionalised and functionalised nanoparticles in their ability to segregate different but closely related cell phenotypes. The results indicate that functionalised gold nanoparticles are more efficient in distinguishing between different types of cells. Our studies pave the way for understanding the uptake of gold nanoparticles and their utilisation for SERS to give rise to a greater biochemical understanding in cell-based therapies.

Infrared microspectroscopy identifies biomolecular changes associated with chronic oxidative stress in mammary epithelium and stroma of breast tissues from healthy young women: implications for latent stages of breast carcinogenesis.

Studies of the decades-long latent stages of breast carcinogenesis have been limited to when hyperplastic lesions are already present. Investigations of earlier stages of breast cancer (BC) latency have been stymied by the lack of fiducial biomarkers needed to identify where in histologically normal tissues progression toward a BC might be taking place. Recent evidence suggests that a marker of chronic oxidative stress (OxS), protein adducts of 4-hydroxy-2-nonenal (4HNE), can meet this need. Specifically: (1) 4HNE immunopositive (4HNE+) mammary epithelial (ME) cells were found to be prevalent in normal (reduction mammoplasty) tissues of most women (including many teenagers) studied, representative of those living in the United States' high risk-posing environment and: (2) marked (> 1.5-fold) differences were identified between tissues of healthy young women with many vs. few 4HNE+ ME cells in the relative levels of transcripts for 42 of the 84 OxS-associated genes represented in SABioscience Oxidative-Stress/Oxidative-Defense PCR array. Herein we used synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to identify molecular changes associated with 4HNE adducts in basal and luminal ME cells in terminal ductal units (TDLU), which are the cells of origin of BC, and associated intralobular and interlobular stroma, known contributors to carcinogenesis. Multivariate analysis-derived wavenumbers differentiated 4HNE+ and 4HNE- cells in each of the anatomical compartments. Specifically, principal component and linear discriminant analyses of mid-infrared spectra obtained from these cells revealed unambiguous, statistically highly significant differences in the "biochemical fingerprint" of 4HNE+ vs. 4HNE- luminal and basal ME cells, as well as between associated intralobular and interlobular stroma. These findings demonstrate further SR-FTIR microspectroscopy's ability to identify molecular changes associated with altered physiological and/or pathophysiological states, in this case with a state of chronic OxS that provides a pro-carcinogenic microenvironment.

CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells.

Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo.

Bronchodilator, vasodilator and spasmolytic activities of Cymbopogon martinii.

Cymbopogon martinii (Cm.Cr) is traditionally used in south Asian communities for the management of multiple ailments including gastrointestinal, respiratory and vascular disorders and the present study was undertaken to validate these folkloric uses. The application of a methanol extract of the plant (Cm.Cr) to isolated rabbit jejunum preparation exhibited relaxation through decrease in magnitude and frequency of spontaneous contractions. The Cm.Cr also exerted relaxant effect on high K(+) (80 mM) induced contractions in isolated rabbit jejunum preparations. The Cm.Cr and its dichloromethane (Cm.Dcm) and aqueous (Cm.Aq) fractions also caused concentration-dependent relaxation in spontaneous and K(+) (80 mM) induced contractions which are comparables to effects produced by verapamil. Cm.Cr caused shifting of the Ca(2+)-curves toward right, suggesting the presence of a Ca(2+) channel blocking activity. Subsequently, Cm.Cr, Cm.Dcm and Cm.Aq caused relaxation of CCh (1 µM) and K(+) (80 mM) induced contractions in isolated rabbit tracheal preparations, suggesting that the observed relaxant effect can be mediated through antimuscarinic and/or Ca(2+) channel blocking activities. Cm.Cr tested against phenylephrine (PE; 1 µM) and K(+) (80 mM) induced contractions exhibited partial relaxation of isolated rabbit aortic preparations. The above-mentioned studies provided a scientific basis for the folkloric use of Cymbopogon martini in the management of multiple ailments in traditional systems of medicines.

Exploiting biospectroscopy as a novel screening tool for cervical cancer: towards a framework to validate its accuracy in a routine clinical setting.

Biospectroscopy is an emerging field that harnesses the platform of physical sciences with computational analysis in order to shed novel insights on biological questions. An area where this approach seems to have potential is in screening or diagnostic clinical settings, where there is an urgent need for new approaches to objectively interrogate large numbers of samples in an objective fashion with acceptable levels of sensitivity and specificity. This review outlines the benefits of biospectroscopy in screening for precancer lesions of the cervix due to its ability to separate different grades of dysplasia. It evaluates the feasibility of introducing this technique into cervical screening programs on the basis of its ability to identify biomarkers of progression within derived spectra ('biochemical­cell fingerprints').

Bronchodilator, vasodilator and spasmolytic activities of methanolic extract of Myrtus communis L.

The present study was undertaken to validate some of the folkloric claims about the effectiveness of the use of a Myrtus communis L. crude methanol extract (Mc.Cr) in gastrointestinal, respiratory and vascular diseases. Mc.Cr caused complete relaxation of spontaneous and K⁺ (80 mM)-induced contractions in isolated rabbit jejunum. It caused right ward parallel shift of calcium concentration response curves. Mc.Cr exhibited relaxant effect on CCh- and K⁺ (80 mM)-induced contractions in isolated rabbit tracheal preparations. Furthermore, Mc.Cr caused relaxation of phenylephrine (1 µM)- and K⁺ (80 mM)-induced contractions in isolated rabbit aorta preparations. These effects were similar to verapamil, a standard calcium channel blocker. These findings could be the basis for explaining the spasmolytic, bronchodilator and vasodilator activities of the extract, through a possible calcium channel blocking activity.

Biospectroscopy insights into the multi-stage process of cervical cancer development: probing for spectral biomarkers in cytology to distinguish grades.

Cervical cancer screening programmes have greatly reduced the burden associated with this disease. However, conventional cervical cytology screening still lacks sensitivity and specificity. There is an urgent need for the development of a low-cost robust screening technique. By generating a spectral "biochemical-cell fingerprint", Fourier-transform infrared (FTIR) spectroscopy has been touted as a tool capable of segregating grades of dysplasia. A total of 529 specimens were collected over a period of one year at two colposcopy centres in Dublin, Ireland. Of these, n = 128 were conventionally classed as high-grade, n = 186 as low-grade and n = 215 as normal. Following FTIR spectroscopy, derived spectra were examined for segregation between classes in scores plots generated with subsequent multivariate analysis. A degree of crossover between classes was noted and this could be associated with imperfect conventional screening resulting in an inaccurate diagnosis or an incomplete transition between classes. Maximal crossover associated with n = 102 of 390 specimens analyzed was found between normal and low-grade specimens. However, robust spectral differences (P≤ 0.0001) were still observed at 1512 cm(-1), 1331 cm(-1) and 937 cm(-1). For high-grade vs. low-grade specimens, spectral differences (P≤ 0.0001) were observed at Amide I (1624 cm(-1)), Amide II (1551 cm(-1)) and asymmetric phosphate stretching vibrations (νasPO2(-); 1215 cm(-1)). Least crossover (n = 50 of 343 specimens analyzed) was seen when comparing high-grade vs. normal specimens; significant inter-class spectral differences (P≤ 0.0001) were noted at Amide II (1547 cm(-1)), 1400 cm(-1) and 995 cm(-1). Deeper understanding of the underlying changes in the transition between cervical cytology classes (normal vs. low-grade vs. high-grade) is required in order to develop biospectroscopy tools as a screening approach. This will then allow for the development of blind classification algorithms.

Sub-cellular spectrochemical imaging of isolated human corneal cells employing synchrotron radiation-based Fourier-transform infrared microspectroscopy.

Understanding stem cell (SC) biology remains challenging and one of the few human tissues within which their in situ location is well characterized is the cornea. Individual human corneal epithelial cells were isolated from biopsies of live tissues using fluorescence-activated cell sorting (FACS); these were divided into putative SCs, transit-amplifying (TA) cells and terminally-differentiated (TD) cells. Employing synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy with a focal plane array (FPA), sub-cellular spatial resolution analysis of unstained isolated cells was achieved as a consequence of the brilliance of a 12 collimated beams arrangement allowing rapid spectral acquisition. Infrared (IR) spectra were extracted and pre-processed. Subsequent categorization with multivariate analysis of IR spectra derived from FPA images was used to investigate biomolecular changes between classes. A progressive segregation in cell-specific spectral categories with differentiation from SC to TA cell to TD cell was noted. Multiple different absorption peaks that discriminated putative SCs, TA cells and TD cells across DNA, protein and lipid spectral regions were identified. DNA regions (1080 and 1225 cm(-1)) and some protein regions (1443 cm(-1)) primarily segregated SCs from TA cells and TD cells, whilst amide regions and lipids (1,550, 1650 and 1740 cm(-1)) segregated TA cells and TD cells. Scanning electron microscopy images verified the external phenotypic characteristics of the different isolated cell types. These findings highlight the applicability of SR-FTIR microspectroscopy towards distinguishing SCs, TA cells and TD cells, and suggest that cellular classification via traditional methods of immunolabelling can be greatly aided by the use of spectral biomarkers.