Inside Front Cover: Combining hyperspectral imaging and chemometrics to assess and interpret the effects of environmental stressors on zebrafish eye images at tissue level (J. Biophotonics 3/2018)

Raman images were used to study the effect of the contaminant chlorpyriphos‐oxon on zebrafish eye samples. Multivariate Curve Resolution‐Alternating Least Squares (MCR‐ALS) was used to obtain the distribution maps and spectral signatures of biological components present in the images analyzed. The use of MCRALS spectral signatures as starting information for Partial Least Squares‐Discriminant Analysis allowed statistical assessment of the effect of the contaminant at a specific tissue level. Further details can be found in the article by Víctor Olmos et al. ( e201700089 ).

     

Diagnosis of early gastric cancer based on fluorescence hyperspectral imaging technology combined with partial‐least‐square discriminant analysis and support vector machine

This study investigated the feasibility of using fluorescence hyperspectral imaging technology to diagnose of early‐stage gastric cancer. Fluorescence spectral images of 76 patients who were pathologically diagnosed as non‐atrophic gastritis, premalignant lesions and gastric cancer were collected. Fluorescence spectra at 100‐pixel points were randomly extracted after binarization. Diagnostic models of non‐atrophic gastritis, premalignant lesions and gastric cancer were constructed through partial‐least‐square discriminant analysis (PLS‐DA) and support vector machine (SVM) algorithms. The prediction effects of PLS‐DA and SVM models were compared. Results showed that the average spectra of normal, precancerous and gastric cancer tissues significantly differed at 496, 546, 640 and 670 nm, and regular changes in fluorescence intensity at 546 nm were in the following order: normal > precancerous lesions > gastric cancer. Additionally, the effect of the diagnostic model established by SVM is significantly better than PLS‐DA which accuracy, specificity and sensitivity are above 94%. Experimental results revealed that the fast diagnostic model of early gastric cancer by combining fluorescence hyperspectral imaging technology and improved SVM was effective and feasible, thereby providing an accurate and rapid method for diagnosing early‐stage gastric cancer.      

Determinants of buildup of the toxic dopamine metabolite DOPAL in Parkinson's disease

Intra‐neuronal metabolism of dopamine (DA) begins with production of 3,4‐dihydroxyphenylacetaldehyde (DOPAL), which is toxic. According to the ‘catecholaldehyde hypothesis,’ DOPAL destroys nigrostriatal DA terminals and contributes to the profound putamen DA deficiency that characterizes Parkinson's disease (PD). We tested the feasibility of using post‐mortem patterns of putamen tissue catechols to examine contributions of altered activities of the type 2 vesicular monoamine transporter (VMAT2) and aldehyde dehydrogenase (ALDH) to the increased DOPAL levels found in PD. Theoretically, the DA : DOPA concentration ratio indicates vesicular uptake, and the 3,4‐dihydroxyphenylacetic acid : DOPAL ratio indicates ALDH activity. We validated these indices in transgenic mice with very low vesicular uptake (VMAT2‐Lo) or with knockouts of the genes encoding ALDH1A1 and ALDH2 (ALDH1A1,2 KO), applied these indices in PD putamen, and estimated the percent decreases in vesicular uptake and ALDH activity in PD. VMAT2‐Lo mice had markedly decreased DA:DOPA (50 vs. 1377, p < 0.0001), and ALDH1A1,2 KO mice had decreased 3,4‐dihydroxyphenylacetic acid:DOPAL (1.0 vs. 11.2, p < 0.0001). In PD putamen, vesicular uptake was estimated to be decreased by 89% and ALDH activity by 70%. Elevated DOPAL levels in PD putamen reflect a combination of decreased vesicular uptake of cytosolic DA and decreased DOPAL detoxification by ALDH.

     

Investigation of human pancreatic cancer tissues by Fourier Transform Infrared Hyperspectral Imaging

Fourier‐transform infrared hyperspectral imaging (FTIR‐HSI) provides hyperspectral images containing both morphological and chemical information. It is widely applied in the biomedical field to detect tumor lesions, even at the early stage, by identifying specific spectral biomarkers. Pancreatic neoplasms present different prognoses and are not always easily classified by conventional analyses. In this study, tissue samples with diagnosis of pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor were analyzed by FTIR‐HSI and the spectral data compared with those from healthy and dysplastic samples. Multivariate/univariate approaches were complemented to hyperspectral images, and definite spectral markers of the different lesions identified. The malignant lesions were recognizable both from healthy/dysplastic pancreatic tissues (high values of phospholipids and triglycerides with shorter, more branched and less unsaturated alkyl chains) and between each other (different amounts of total lipids, phosphates and carbohydrates). These findings highlight different metabolic pathways characterizing the different samples, well detectable by FTIR‐HSI.     

Optogenetic versus electrical stimulation of dopamine terminals in the nucleus accumbens reveals local modulation of presynaptic release

The nucleus accumbens is highly heterogeneous, integrating regionally distinct afferent projections and accumbal interneurons, resulting in diverse local microenvironments. Dopamine (DA) neuron terminals similarly express a heterogeneous collection of terminal receptors that modulate DA signaling. Cyclic voltammetry is often used to probe DA terminal dynamics in brain slice preparations; however, this method traditionally requires electrical stimulation to induce DA release. Electrical stimulation excites all of the neuronal processes in the stimulation field, potentially introducing simultaneous, multi‐synaptic modulation of DA terminal release. We used optogenetics to selectively stimulate DA terminals and used voltammetry to compare DA responses from electrical and optical stimulation of the same area of tissue around a recording electrode. We found that with multiple pulse stimulation trains, optically stimulated DA release increasingly exceeded that of electrical stimulation. Furthermore, electrical stimulation produced inhibition of DA release across longer duration stimulations. The GABA_B antagonist, CGP 55845, increased electrically stimulated DA release significantly more than light stimulated release. The nicotinic acetylcholine receptor antagonist, dihydro‐β‐erythroidine hydrobromide, inhibited single pulse electrically stimulated DA release while having no effect on optically stimulated DA release. Our results demonstrate that electrical stimulation introduces local multi‐synaptic modulation of DA release that is absent with optogenetically targeted stimulation.

     

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

We demonstrate a novel bio‐spectroscopic technique, “simultaneous Raman/GFP microspectroscopy”. It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti‐Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two‐component multivariate curve resolution with alternate least squares (MCR‐ALS) analysis in the anti‐Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR‐ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm^?1, previously reported as “Raman spectroscopic signature of life of yeast.”     

Induction of depressive‐like effects by subchronic exposure to cocaine or heroin in laboratory rats

The effect of psychoactive drugs on depression has usually been studied in cases of prolonged drug addiction and/or withdrawal, without much emphasis on the effects of subchronic or recreational drug use. To address this issue, we exposed laboratory rats to subchronic regimens of heroin or cocaine and tested long‐term effects on (i) depressive‐like behaviors, (ii) brain‐derived neurotrophic factor (BDNF) levels in reward‐related brain regions, and (iii) depressive‐like behavior following an additional chronic mild stress procedure. The long‐term effect of subchronic cocaine exposure was a general reduction in locomotor activity whereas heroin exposure induced a more specific increase in immobility during the forced swim test. Both cocaine and heroin exposure induced alterations in BDNF levels that are similar to those observed in several animal models of depression. Finally, both cocaine and heroin exposure significantly enhanced the anhedonic effect of chronic mild stress. These results suggest that subchronic drug exposure induces depressive‐like behavior which is accompanied by modifications in BDNF expression and increases the vulnerability to develop depressive‐like behavior following chronic stress. Implications for recreational and small‐scale drug users are discussed.

     

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca²⁺ homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca²⁺ entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L‐ and T‐type voltage‐gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca²⁺ and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca²⁺ overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA‐binding proteins transactive response DNA‐binding protein 43 (TDP‐43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP‐43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

     

Epidermal fatty acid‐binding protein protects nerve growth factor‐differentiated PC12 cells from lipotoxic injury

Epidermal fatty acid‐binding protein (E‐FABP/FABP5/DA11) binds and transport long‐chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E‐FABP protects nerve growth factor‐differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM‐induced lipotoxicity (PAM‐LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E‐FABP. Antioxidants MCI‐186 and N‐acetyl cysteine prevented E‐FABP's induction in expression by PAM‐LTx, while tert‐butyl hydroperoxide increased ROS and E‐FABP expression. Non‐metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E‐FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE‐FABP showed reduced E‐FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E‐FABP cellular levels by pre‐loading the cells with recombinant E‐FABP diminished the PAM‐induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E‐FABP expression and enhanced the resistance of NGFDPC12 cells to PAM‐LTx. We conclude that E‐FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS.

     

Dysregulated molecular pathways in amyotrophic lateral sclerosis–frontotemporal dementia spectrum disorder

Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72. As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALS‐FTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre‐mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders.     

Biochemical characterization of metabolism‐based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F₂ population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F₂ population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.     

Chemical Composition,Antibacterial, Schistosomicidal,and Cytotoxic Activities of the Essential Oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae)

We have investigated the chemical composition and the antibacterial activity of the essential oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae) (DA‐EO) against a representative panel of cariogenic bacteria. We have also assessed the in vitro schistosomicidal effects of DA‐EO on Schistosoma mansoni and its cytotoxicity to GM07492‐A cells in vitro. Gas chromatography (GC) and gas chromatography‐mass spectrometry (GC/MS) revealed that the monoterpenes cis‐piperitone oxide (35.2%), p‐cymene (14.5%), isoascaridole (14.1%), and α‐terpinene (11.6%) were identified by as the major constituents of DA‐EO. DA‐EO displayed weak activity against Streptococcus sobrinus and Enterococcus faecalis (minimum inhibitory concentration (MIC) = 1000 μg/ml). On the other hand, DA‐EO at 25 and 12.5 μg/ml presented remarkable schistosomicidal action in vitro and killed 100% of adult worm pairs within 24 and 72 h, respectively. The LC₅₀ values of DA‐EO were 6.50 ± 0.38, 3.66 ± 1.06, and 3.65 ± 0.76 μg/ml at 24, 48, and 72 h, respectively. However, DA‐EO at concentrations higher than 312.5 μg/ml significantly reduced the viability of GM07492‐A cells (IC₅₀ = 207.1 ± 4.4 μg/ml). The selectivity index showed that DA‐EO was 31.8 times more toxic to the adult S. mansoni worms than GM07492‐A cells. Taken together, these results demonstrate the promising schistosomicidal potential of the essential oil of Dysphania ambrosioides.     

Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High‐throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo‐response. This study was aimed to differentiate gemcitabine‐sensitive and gemcitabine‐resistant induction in two CCA cell lines (KKU‐M139 and KKU‐M214) and xenograft tissues using synchrotron‐FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS‐DA) could discriminate between chemo‐sensitive and chemo‐resistant cells in the FTIR fingerprint spectral region (1800–1000 cm^–1) with more than 90% sensitivity and specificity. The chemo‐resistant and chemo‐sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH₃ deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo‐resistant and chemo‐sensitive CCA tissues, and PLS‐DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo‐response both in vitro and in vivo.

     

Optical sensory arrays for the detection of urinary bladder cancer‐related volatile organic compounds

Non‐invasive detection of urinary bladder cancer remains a significant challenge. Urinary volatile organic compounds (VOCs) are a promising alternative to cell‐based biomarkers. Herein, we demonstrate a novel diagnosis system based on an optic fluorescence sensor array for detecting urinary bladder cancer VOCs biomarkers. This study describes a fluorescence‐based VOCs sensor array detecting system in detail. The choice of VOCs for the initial part was based on an extensive systematic search of the literature and then followed up using urinary samples from patients with urinary bladder transitional cell carcinoma. Canonical discriminant analysis and partial least squares discriminant analysis (PLS‐DA) were employed and correctly detected 31/48 urinary bladder cancer VOC biomarkers and achieved an overall 77.75% sensitivity and 93.25% specificity by PLS‐DA modelling. All five urine samples from bladder cancer patients, and five healthy controls were successfully identified with the same sensor arrays. Overall, the experiments in this study describe a real‐time platform for non‐invasive bladder cancer diagnosis using fluorescence‐based gas‐sensor arrays. Pure VOCs and urine samples from the patients proved such a system to be promising; however, further research is required using a larger population sample.      

Intraoperative multispectral and hyperspectral label‐free imaging: A systematic review of in vivo clinical studies

Multispectral and hyperspectral imaging (HSI) are emerging optical imaging techniques with the potential to transform the way surgery is performed but it is not clear whether current systems are capable of delivering real‐time tissue characterization and surgical guidance. We conducted a systematic review of surgical in vivo label‐free multispectral and HSI systems that have been assessed intraoperatively in adult patients, published over a 10‐year period to May 2018. We analysed 14 studies including 8 different HSI systems. Current in‐vivo HSI systems generate an intraoperative tissue oxygenation map or enable tumour detection. Intraoperative tissue oxygenation measurements may help to predict those patients at risk of postoperative complications and in‐vivo intraoperative tissue characterization may be performed with high specificity and sensitivity. All systems utilized a line‐scanning or wavelength‐scanning method but the spectral range and number of spectral bands employed varied significantly between studies and according to the system's clinical aim. The time to acquire a hyperspectral cube dataset ranged between 5 and 30 seconds. No safety concerns were reported in any studies. A small number of studies have demonstrated the capabilities of intraoperative in‐vivo label‐free HSI but further work is needed to fully integrate it into the current surgical workflow.      

Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness

Gulf War Illness (GWI) is a multi‐symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N‐diethyl‐meta‐toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti‐inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7–14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain‐wide neuroinflammation assessed by qPCR of tumor necrosis factor‐α, IL6, chemokine (C‐C motif) ligand 2, IL‐1β, leukemia inhibitory factor, and oncostatin M. Pre‐treatment with high physiological levels of CORT greatly augmented (up to 300‐fold) the neuroinflammatory responses to DFP. Anti‐inflammatory pre‐treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI.

     

Vinpocetine and α‐tocopherol prevent the increase in DA and oxidative stress induced by 3‐NPA in striatum isolated nerve endings

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3‐nitropropionic acid (3‐NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum‐isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4‐dihydroxyphenylacetic acid (DOPAC), and that 3‐NPA increased DA, reactive oxygen species (ROS), DA‐quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3‐NPA‐induced increase in DA, ROS, lipid peroxidation, and DA‐quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α‐tocopherol. Results show that the increase in DA induced by 3‐NPA was inhibited by both 25 μM vinpocetine and 50 μM α‐tocopherol. Vinpocetine, as α‐tocopherol, also inhibited 3‐NPA‐induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA‐RS formation), and DA‐quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3‐NPA, 3‐NPA increases DA‐oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α‐tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA‐rich structures.