Spatial Elucidation of Spinal Cord Lipid- and Metabolite-Regulations in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressing disease of the central nervous system that is characterized by motor neuron degeneration in the brain stem and the spinal cord. We employed time of flight secondary ion mass spectrometry (ToF-SIMS) to profile spatial lipid-and metabolite-regulations in post mortem human spinal cord tissue from ALS patients to investigate chemical markers of ALS pathogenesis. ToF-SIMS scans and multivariate analysis of image and spectral data were performed on thoracic human spinal cord sections. Multivariate statistics of the image data allowed delineation of anatomical regions of interest based on their chemical identity. Spectral data extracted from these regions were compared using two different approaches for multivariate statistics, for investigating ALS related lipid and metabolite changes. The results show a significant decrease for cholesterol, triglycerides, and vitamin E in the ventral horn of ALS samples, which is presumably a consequence of motor neuron degeneration. Conversely, the biogenic mediator lipid lysophosphatidylcholine and its fragments were increased in ALS ventral spinal cord, pointing towards neuroinflammatory mechanisms associated with neuronal cell death. ToF-SIMS imaging is a promising approach for chemical histology and pathology for investigating the subcellular mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis

MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and small molecules within biological systems through the in situ analysis of tissue sections. MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement and enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. In recent years, a great many advances in the practice of imaging mass spectrometry have taken place, making the technique more sensitive, robust, and ultimately useful. In this review, we focus on the current state of the art of MALDI-IMS, describe basic technological developments for MALDI-IMS of animal and human tissues, and discuss some recent applications in basic research and in clinical settings

Localization of Secondary Metabolites in Marine Invertebrates: Contribution of MALDI MSI for the Study of Saponins in Cuvierian Tubules of H. forskali

BACKGROUND: Several species of sea cucumbers of the family Holothuriidae possess a particular mechanical defense system called the Cuvierian tubules (Ct). It is also a chemical defense system as triterpene glycosides (saponins) appear to be particularly concentrated in Ct. In the present study, the precise localization of saponins in the Ct of Holothuria forskali is investigated. Classical histochemical labeling using lectin was firstly performed but did not generate any conclusive results. Thus, MALDI mass spectrometry Imaging (MALDI-MSI) was directly applied and completed by statistical multivariate tests. A comparison between the tubules of relaxed and stressed animals was realized. RESULTS: These analyses allowed the detection of three groups of ions, corresponding to the isomeric saponins of the tubules. Saponins detected at m/z 1287 and 1303 were the most abundant and were apparently localized in the connective tissue of the tubules of both relaxed and stressed individuals. Saponins at m/z 1125 and 1141 were detected in lower amount and were present in tissues of relaxed animals. Finally, saponin ions at 1433, 1449, 1463 and 1479 were observed in some Ct of stressed holothuroids in the outer part of the connective tissue. The saponin group m/z 14xx seems therefore to be stress-specific and could originate from modifications of the saponins with m/z of 11xx. CONCLUSIONS: All the results taken together indicate a complex chemical defense mechanism with, for a single organ, different sets of saponins originating from different cell populations and presenting different responses to stress. The present study also reflects that MALDI-MSI is a valuable tool for chemical ecology studies in which specific chemical signalling molecules like allelochemicals or pheromones have to be tracked. This report represents one of the very first studies using these tools to provide a functional and ecological understanding of the role of natural products from marine invertebrates

Multivariate analyses for biomarkers hunting and validation through on-tissue bottom-up or in-source decay in MALDI-MSI: application to prostate cancer.

peer reviewe

Role of Optical Spectroscopic Methods in Neuro-Oncological Sciences

In the surgical treatment of malignant tumors, it is crucial to characterize the tumor as precisely as possible. The determination of the exact tumor location as well as the analysis of its properties is very important in order to obtain an accurate diagnosis as early as possible. In neurosurgical applications, the optical, non-invasive and in situ techniques allow for the label-free analysis of tissue, which is helpful in neuropathology. In the past decades, optical spectroscopic methods have been investigated drastically in the management of cancer. In the optical spectroscopic techniques, tissue interrogate with sources of light which are ranged from the ultraviolet to the infrared wavelength in the spectrum. The information accumulation of light can be in a reflection which is named reflectance spectroscopy; or interactions with tissue at different wavelengths which are called fluorescence and Raman spectroscopy. This review paper introduces the optical spectroscopic methods which are used to characterize brain tumors (neuro-oncology). Based on biochemical information obtained from these spectroscopic methods, it is possible to identify tumor from normal brain tissues, to indicate tumor margins, the borders towards normal brain tissue and infiltrating gliomas, to distinguish radiation damage of tissues, to detect particular central nervous system (CNS) structures to identify cell types using particular neurotransmitters, to detect cells or drugs which are optically labeled within therapeutic intermediations and to estimate the viability of tissue and the prediction of apoptosis beginning in vitro and in vivo. The label-free, optical biochemical spectroscopic methods can provide clinically relevant information and need to be further exploited to develop a safe and easy-to-use technology for in situ diagnosis of malignant tumors

Normalization in MALDI-TOF imaging datasets of proteins: practical considerations

Normalization is critically important for the proper interpretation of matrix-assisted laser desorption/ionization (MALDI) imaging datasets. The effects of the commonly used normalization techniques based on total ion count (TIC) or vector norm normalization are significant, and they are frequently beneficial. In certain cases, however, these normalization algorithms may produce misleading results and possibly lead to wrong conclusions, e.g. regarding to potential biomarker distributions. This is typical for tissues in which signals of prominent abundance are present in confined areas, such as insulin in the pancreas or β-amyloid peptides in the brain. In this work, we investigated whether normalization can be improved if dominant signals are excluded from the calculation. Because manual interaction with the data (e.g., defining the abundant signals) is not desired for routine analysis, we investigated two alternatives: normalization on the spectra noise level or on the median of signal intensities in the spectrum. Normalization on the median and the noise level was found to be significantly more robust against artifact generation compared to normalization on the TIC. Therefore, we propose to include these normalization methods in the standard “toolbox” of MALDI imaging for reliable results under conditions of automation

Incorporating standardised drift-tube ion mobility to enhance non-targeted assessment of the wine metabolome (LC×IM-MS)

Liquid chromatography with drift-tube ion mobility spectrometry-mass spectrometry (LCxIM-MS) is emerging as a powerful addition to existing LC-MS workflows for addressing a diverse range of metabolomics-related questions [1,2]. Importantly, excellent precision under repeatability and reproducibility conditions of drift-tube IM separations [3] supports the development of non-targeted approaches for complex metabolome assessment such as wine characterisation [4]. In this work, fundamentals of this new analytical metabolomics approach are introduced and application to the analysis of 90 authentic red and white wine samples originating from Macedonia is presented. Following measurements, intersample alignment of metabolites using non-targeted extraction and three-dimensional alignment of molecular features (retention time, collision cross section, and high-resolution mass spectra) provides confidence for metabolite identity confirmation. Applying a fingerprinting metabolomics workflow allows statistical assessment of the influence of geographic region, variety, and age. This approach is a state-of-the-art tool to assess wine chemodiversity and is particularly beneficial for the discovery of wine biomarkers and establishing product authenticity based on development of fingerprint libraries

Exploration of urological biomarkers by urine metabolome NMR-analysis in an Asian patient cohort of prostate cancer

1.Prostate Cancer (PC) 1.1.Epidemiology Prostate cancer (PC) is one of the major threats to men’s health worldwide (Siegel et al., 2016; Brawley, 2012; Jahn et al., 2015; Center et al., 2012). In the United States PC was estimated to make up roughly 20% of the new cancer cases in men in 2016. Deaths from PC are expected to account for 8% of cancer associated deaths (Siegel et al., 2016). Epidemiological data from China are still rare and incomplete but were recently supplemented by high-quality data provided by the National Central Cancer Registry of China (NCCR) (Chen et al., 2016). The incidence rate of prostate cancer in China increased from 1998 to 2008 by a factor of 3, from 35.2/100,000 to 110.0/100,000 and the average annual growth rate was as high as 12.07% reaching 60,300 cases in 2015 (Zhu et al., 2015; Coffey, 2001; Baade et al., 2013; Chen et al., 2016). While incidence rates in rural areas remained stable between 2006 and 2009, there was an increase in urban areas, especially documented in Hong Kong and Shanghai. The rapid rise of the incidence rate may be in part related to the aging of the population but there seems to be a strong link to Western-style diet (Lin et al., 2015). A comparison of the incidences of prostate cancer in 2015 showed that although the total number of patients in the United States has reached 3.66 times that of China, the estimated death tolls in the two countries are almost similar (Table 1) (Siegel et al., 2016; Ervik et al., 2016; Chen et al., 2016).Interestingly, the numbers in the European Union (EU, WHO region) are in between which might reflect more regional variations in living conditions and diet. However, further investigations are required to come to valid conclusions. Effectivity of PC treatment and cancer recurrence heavily depend on early detection and proper risk stratification (Moller et al., 2015; Schroder et al., 2012; Klotz et al., 2015; Moyer, 2012). In the US, the proportion of localized prostate cancer accounts for more than 80% of all cases, which is also one of the major reasons the mortality/morbidity rate in the US is much lower than that in Asian countries, and continues to decrease (Moller et al., 2015; Jemal et al., 2015; DeSantis et al., 2014). Therefore, early detection and diagnosis is the most effective way by which to improve the survival rate, and development of new biomarkers and/or reasonable combination of current diagnostic methods is a hot spot in the field of prostate cancer research (Felgueiras et al., 2014). Among countries that have implemented prostate cancer screening strategies, five-year survival rates have improved rapidly in Japan, with an average annual increase of about 11.7% and a 5-year survival rate of 93%, while in China, the annual increase was only 3.7% and the 5-year survival rate was 69.2%(Yao et al., 2021). In 2018, there were 1.3 million new cases of prostate cancer worldwide, and its morbidity and mortality ranked second and fifth among male malignancies, respectively.However, to date no serum or urine biomarker or biomarker panel meets the requirements for highly sensitive and specific detection of PC and differentiation between indolent and significant PC. We here explore the prospects of metabolomics to improve prostate cancer detection, patient stratification and treatment monitoring. 1.2. PC classification and grading The prostate gland is a walnut-sized gland located between the bladder neck and the external urethral sphincter. There are four main zones in the prostate gland: the peripheral zone (posteriorly), the fibromuscular zone (anteriorly), the central zone (centrally) and the transitional zone (surrounding the urethra). The anatomy of the prostate gland is shown in Fig. 1 (Adapted from: Bhavsar et al., 2014).Prostate cancer does not occur uniformly throughout the prostate. Although cancers of the prostate often are multifocal, from 80% to 85% arise from the peripheral zone, 10% to 15% arise from the transition zone, and 5% to 10% arise from the central zone (Buyyounouski et al., 2017). The biopsy Gleason grading system is the most important prognostic marker for prostate cancer. The higher the Gleason score, the higher the malignant degree of prostate cancer. The TNM staging system proposed by AJCC is a widely used independent index that can reflect the progression and prognosis of prostate cancer. Table 2 shows the definitions for clinical and pathological T, N, and M classifications (Buyyounouski et al., 2017). Radical prostatectomy (RP) has become the most effective method for the treatment of localized prostate cancer and some high-risk prostate cancer. RP is used when the cancer is believed to be confined to the prostate gland. During the procedure, the prostate gland and some tissue around the gland, including the seminal vesicles, are removed. Transurethral resection of the prostate, or TURP, which also involves removal of part of the prostate gland, is an approach performed through the penis with an endoscope (small, flexible tube with a light and a lens on the end). This procedure doesn't cure prostate cancer but can remove the obstruction while the doctors plan for definitive treatment. Laparoscopic surgery, done manually or by robot, is another method of removal of the prostate gland. Shortcomings in comprehensive medical check-ups in low- and middle-income countries lead to delayed detection of PC and are causative of high numbers of advanced PC cases at first diagnosis. The performance of available biomarkers is still insufficient and limited applicability, including logistical and financial burdens, impedes comprehensive implementation into health care systems. There is broad agreement on the need of new biomarkers to improve (i) early detection of PC, (ii) risk stratification, (iii) prognosis, and (iv) treatment monitoring. 2. PC Biomarkers Serum prostate specific antigen (PSA) level and digital rectal examination (DRE) constitute the major screening tests for prostate cancer (PC) diagnosis, while the transrectal ultrasound-guided prostate biopsy provides the final confirmation of cancer presence (Velonas et al., 2013). PSA level has been extensively used as a biomarker to detect PC. Nevertheless, due to prostate physiology, PSA testing results in a large frequency of false positives leading to numerous men each year undergoing unnecessary prostate biopsy procedures (Vickers et al., 2008; Link et al., 2004; McDunn et al., 2013; Roberts et al., 2011; Djavan et al., 2000). Hence, a non-invasive, cost-effective, efficient, and reasonably accurate test for early identification of PC is urgently needed. Compared with serum, urine is easier to obtain and handle, needs less sample preparation, and has higher amounts of metabolites and lower protein content (Rigau et al., 2013; Wilkosz et al., 2011; Zhang et al., 2013). Therefore, in attempt to solve this diagnostic dilemma, many previous studies have focused on urinary metabolomic profile, to identify the predictive biomarkers for PC (Chistiakov et al., 2018). Yang and colleagues conducted a study searching for urine metabolite biomarkers for the detection of PC. They found twenty differentially expressed urine metabolites in a cohort of 50 prostate cancer patients compared to non-cancerous individuals (Yang et al., 2021; Gordetsky et al., 2016; Nam et al., 2018; Di Meo et al., 2017). The combination of solely three metabolites, representing alterations in Glycine, Serine, and Threonine metabolism (KEGG database pathway), was able to identify PC patients with 77% accuracy at 80% sensitivity and 64% specificity. Furthermore, those metabolites could separate significant PC (Gleason score ≥ 7) from indolent PC (GS 6), which confirms urine metabolomics as a promising diagnostic tool in PC. However, to date, no single urine biomarker/biomarker panel meets the requirements for highly sensitive, and specific detection of PC. Therefore, the search for PC-specific biomarkers still is an active area of research. 3. PC prevalence is not equal in different populations There is a racial difference in incidence rate and interpatient heterogeneity of prostate cancer. By contrast, Asian men have lower disease prevalence compared with Asian-American or American PC cohorts. Despite lower PC incidence, the Asian populations have a higher prevalence of advanced disease, probably due to the lack of availability of more sensitive diagnostic tools (Ateeq et al., 2016). Therefore, it’s necessary to define the urine metabolome in an Asian population. 4. Aims of the study − Exploration of novel biomarkers for the detection of PC in an Asian cohort. − Are urinary metabolomics suitable to develop new PC biomarkers? − What are the advantages of urine biomarkers? − How to identify novel biomarkers in the urine and to investigate the possible functions and roles of potential biomarkers in PC