Metabolic Basis of Circadian Dysfunction in Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. The management of PD is a challenging aspect for general physicians and neurologists. It is characterized by the progressive loss of dopaminergic neurons. Impaired α-synuclein secretion and dopamine release may cause mitochondrial dysfunction and perturb energy metabolism, subsequently altering the activity and survival of dopaminergic neurons, thus perpetuating the neurodegenerative process in PD. While the etiology of PD remains multifactorial, emerging research indicates a crucial role of circadian dysfunction in its pathogenesis. Researchers have revealed that circadian dysfunction and sleep disorders are common among PD subjects and disruption of circadian rhythms can increase the risk of PD. Hence, understanding the findings of circadian biology from translational research in PD is important for reducing the risk of neurodegeneration and for improving the quality of life. In this review, we discuss the intricate relationship between circadian dysfunction in cellular metabolism and PD by summarizing the evidence from animal models and human studies. Understanding the metabolic basis of circadian dysfunction in PD may shed light on novel therapeutic approaches to restore circadian rhythm, preserve dopaminergic function, and ameliorate disease progression. Further investigation into the complex interplay between circadian rhythm and PD pathogenesis is essential for the development of targeted therapies and interventions to alleviate the burden of this debilitating neurodegenerative disorder.

Rapid metabolic fingerprinting with the aid of chemometric models to identify authenticity of natural medicines: Turmeric, Ocimum, and Withania somnifera study.

Herbal medicines are popular natural medicines that have been used for decades. The use of alternative medicines continues to expand rapidly across the world. The World Health Organization suggests that quality assessment of natural medicines is essential for any therapeutic or health care applications, as their therapeutic potential varies between different geographic origins, plant species, and varieties. Classification of herbal medicines based on a limited number of secondary metabolites is not an ideal approach. Their quality should be considered based on a complete metabolic profile, as their pharmacological activity is not due to a few specific secondary metabolites but rather a larger group of bioactive compounds. A holistic and integrative approach using rapid and nondestructive analytical strategies for the screening of herbal medicines is required for robust characterization. In this study, a rapid and effective quality assessment system for geographical traceability, species, and variety-specific authenticity of the widely used natural medicines turmeric, Ocimum, and Withania somnifera was investigated using Fourier transform near-infrared (FT-NIR) spectroscopy-based metabolic fingerprinting. Four different geographical origins of turmeric, five different Ocimum species, and three different varieties of roots and leaves of Withania somnifera were studied with the aid of machine learning approaches. Extremely good discrimination (R2 > 0.98, Q2 > 0.97, and accuracy = 1.0) with sensitivity and specificity of 100% was achieved using this metabolic fingerprinting strategy. Our study demonstrated that FT-NIR-based rapid metabolic fingerprinting can be used as a robust analytical method to authenticate several important medicinal herbs.

Multi-Omics Reveal Interplay between Circadian Dysfunction and Type2 Diabetes.

Type 2 diabetes is one of the leading threats to human health in the 21st century. It is a metabolic disorder characterized by a dysregulated glucose metabolism resulting from impaired insulin secretion or insulin resistance. More recently, accumulated epidemiological and animal model studies have confirmed that circadian dysfunction caused by shift work, late meal timing, and sleep loss leads to type 2 diabetes. Circadian rhythms, 24-h endogenous biological oscillations, are a fundamental feature of nearly all organisms and control many physiological and cellular functions. In mammals, light synchronizes brain clocks and feeding is a main stimulus that synchronizes the peripheral clocks in metabolic tissues, such as liver, pancreas, muscles, and adipose tissues. Circadian arrhythmia causes the loss of synchrony of the clocks of these metabolic tissues and leads to an impaired pancreas ß-cell metabolism coupled with altered insulin secretion. In addition to these, gut microbes and circadian rhythms are intertwined via metabolic regulation. Omics approaches play a significant role in unraveling how a disrupted circadian metabolism causes type 2 diabetes. In the present review, we emphasize the discoveries of several genes, proteins, and metabolites that contribute to the emergence of type 2 diabetes mellitus (T2D). The implications of these discoveries for comprehending the circadian clock network in T2D may lead to new therapeutic solutions.

Comprehensive Metabolomic Fingerprinting Combined with Chemometrics Identifies Species- and Variety-Specific Variation of Medicinal Herbs: An Ocimum Study.

Identification of plant species is a crucial process in natural products. Ocimum, often referred to as the queen of herbs, is one of the most versatile and globally used medicinal herbs for various health benefits due to it having a wide variety of pharmacological activities. Despite there being significant global demand for this medicinal herb, rapid and comprehensive metabolomic fingerprinting approaches for species- and variety-specific classification are limited. In this study, metabolomic fingerprinting of five Ocimum species (Ocimum basilicum L., Ocimum sanctum L., Ocimum africanum Lour., Ocimum kilimandscharicum Gurke., and Hybrid Tulsi) and their varieties was performed using LC-MS, GC-MS, and the rapid fingerprinting approach FT-NIR combined with chemometrics. The aim was to distinguish the species- and variety-specific variation with a view toward developing a quality assessment of Ocimum species. Discrimination of species and varieties was achieved using principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA), data-driven soft independent modelling of class analogy (DD-SIMCA), random forest, and K-nearest neighbours with specificity of 98% and sensitivity of 99%. Phenolics and flavonoids were found to be major contributing markers for species-specific variation. The present study established comprehensive metabolomic fingerprinting consisting of rapid screening and confirmatory approaches as a highly efficient means to identify the species and variety of Ocimum, being able to be applied for the quality assessment of other natural medicinal herbs.

Superoxide anion radical induced phototoxicity of 2,4,5,6-Tetraminopyrimidine sulfate via mitochondrial-mediated apoptosis in human skin keratinocytes at ambient UVR exposure.

2,4,5,6-Tetraaminopyrimidine sulfate (TAPS) is worldwide the most commonly used developer in hair dyes. As skin is the major organ, which is directly exposed to these permanent hair dyes, a comprehensive dermal safety assessment is needed. Hereto, we studied the photosensitization potential and mechanism involved in dermal phototoxicity of TAPS exposed to the dark and UVA/UVB/Sunlight by using different in-chemico and mammalian (HaCaT) cells, as test systems. Our experimental outcomes illustrate that TAPS get photodegraded (LC-MS/MS) and specifically generated superoxide anion radical (O2•-) under UVA and UVB via type-I photodynamic reaction. The phototoxic potential of TAPS is measured through MTT, NRU, and LDH assays that depicted a significant cell viability reduction at 25 µg/ml concentration and higher. Different cellular stainings (PI uptake, AO/EB, JC-1, NR uptake) suggested the role of mitochondrial-mediated apoptosis. Further, the transcriptomics study revealed upregulation of Apaf-1, Bax, Cytochrome c, Caspase 3, Caspase 9 and downregulation of Catalase and Bcl-2 by TAPS treated cells that strengthen our findings. Thus, the above findings suggest that chronic application of TAPS may be hazardous for human skin and promote various skin diseases.

Elementomics combined with dd-SIMCA and K-NN to identify the geographical origin of rice samples from China, India, and Vietnam.

The COVID-19 pandemic has impacted the food industry and consumers, with production gaps, shipping delays, and changes in supply and demand leading to an increased risk of food fraud. Rice has a high probability for adulteration by food fraudsters, being a staple commodity for more than half the global population, making the assessment of geographical origins of rice for authenticity important in terms of protecting businesses and consumers. In this study, we describe ICP-MS elemental profiling coupled with elementomic modelling to identify the geographical indications of Indian, Chinese, and Vietnamese rice. A PLS-DA model exhibited good discrimination (R2 = 0.8393, Q2 = 0.7673, accuracy = 1.0). Data-driven soft independent modelling of class analogy (dd-SIMCA) and K-nearest neighbours (K-NN) models have good sensitivity (98%) and specificity (100%).

Authentication of organically grown vegetables by the application of ambient mass spectrometry and inductively coupled plasma (ICP) mass spectrometry; The leek case study.

Health conscious and environmentally aware consumers are purchasing more organically produced foods. They prefer organic fruits and leafy vegetables as these are much less likely to have been exposed to contaminants such as pesticides. The detection of fraudulent activity in this area is difficult to undertake, because many chemical plant protection treatments degrade very quickly or can be washed off to remove evidence of their existence. It was found that when combining DART-MS with a compact, inexpensive and robust single quadrupole mass spectrometer, it was possible to differentiate organic from conventional leeks with 93.8% to 100% accuracy. ICP-MS results showed similar performance, with an ability to differentiate conventional from organic leeks with 92.5% to 98.1% accuracy. This study has paved the way for the certification of vegetables as being organically produced. The next step is to create data libraries to support the roll out of the methodologies described.

Rhythmic glucose metabolism regulates the redox circadian clockwork in human red blood cells.

Circadian clocks coordinate mammalian behavior and physiology enabling organisms to anticipate 24-hour cycles. Transcription-translation feedback loops are thought to drive these clocks in most of mammalian cells. However, red blood cells (RBCs), which do not contain a nucleus, and cannot perform transcription or translation, nonetheless exhibit circadian redox rhythms. Here we show human RBCs display circadian regulation of glucose metabolism, which is required to sustain daily redox oscillations. We found daily rhythms of metabolite levels and flux through glycolysis and the pentose phosphate pathway (PPP). We show that inhibition of critical enzymes in either pathway abolished 24-hour rhythms in metabolic flux and redox oscillations, and determined that metabolic oscillations are necessary for redox rhythmicity. Furthermore, metabolic flux rhythms also occur in nucleated cells, and persist when the core transcriptional circadian clockwork is absent in Bmal1 knockouts. Thus, we propose that rhythmic glucose metabolism is an integral process in circadian rhythms.

Food Fingerprinting: Using a Two-Tiered approach to Monitor and Mitigate Food Fraud in Rice.

BACKGROUND: Rice is an important staple food that is consumed around the world. Like many foods, the price of rice varies considerably, from very inexpensive for a low-quality product to premium pricing for highly prized varieties from specific locations. Therefore, like other foods it is vulnerable to economically motivated adulteration through substitution or misrepresentation of inferior-quality rice for more expensive varieties. OBJECTIVE: In this article we describe results of a research project focused on addressing potential food fraud issues related to rice supplies in China, India, Vietnam, and Ghana. Rice fraud manifests differently in each country; therefore, tailored solutions were required. METHOD: Here we describe a two-tiered testing regime of rapid screening using portable Near Infrared technology supported by second tier testing using mass spectrometry-based analysis of suspicious samples. RESULTS: Portable Near Infrared spectroscopy models and laboratory-based Gas Chromatography-Mass Spectrometry methods were developed to differentiate between: high-value Basmati rice varieties and their potential adulterants; six Geographic Indicated protected rice varieties from specific regions within China; various qualities of rice in Ghana and Vietnam; and locally produced and imported rice in Ghana. Furthermore, an Inductively Coupled Plasma-Mass Spectrometry method was developed to support the Chinese rice varieties methods as well as a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry method for quality differentiation in Vietnam. CONCLUSIONS/HIGHLIGHTS: This two-tier approach can provide a substantially increased level of testing through rapid screening outside of the laboratory with the reassurance of corroborating mass spectrometry-based laboratory analysis to support decision making.

Metabolomic fingerprinting of volatile organic compounds for the geographical discrimination of rice samples from China, Vietnam and India.

Rice is one of the most important cereals for human nutrition and is a basic staple food for half of the global population. The assessment of rice geographical origins in terms of its authenticity is of great interest to protect consumers from misleading information and fraud. In the present study, a head space gas chromatography mass spectrometry (HS-GC-MS) strategy for characterising volatile organic compounds (VOCs) profiles to distinguish rice samples from China, India and Vietnam is described. Partial Least Square Discriminant Analysis (PLS-DA) model exhibited a good discrimination (R2 = 0.98182, Q2 = 0.9722, and Accuracy = 1.0) for rice samples from China, India and Vietnam. Moreover, Data-Driven Soft Independent Modelling of Class Analogy (DD-SIMCA) and K-nearest neighbors shown good specificity 100% and accuracy 100% in identifying the origin of samples. The present study established VOC fingerprinting as a highly efficient approach to identify the geographical origin of rice.

Saliva and urine metabolic profiling reveals altered amino acid and energy metabolism in male farmers exposed to pesticides in Madhya Pradesh State, India.

Globally, the human population is exposed to low doses of pesticides due to its extensive use in agriculture. The chronic exposure to pesticides can lead to cancer, depression, anxiety, Parkinson's and Alzheimer's diseases etc. Here, we have made an attempt to use mass spectrometry based metabolomics to investigate the metabolic perturbations induced by the pesticides in the urine and saliva samples of farmers from the Madhya Pradesh State of India. The study was aimed to establish non-invasive matrices like urine and saliva as alternative diagnostic matrices to the occupational exposure studies. Saliva and urine samples were collected from 51 pesticides applicators and acquired metabolic profiles of urine and saliva samples using gas chromatography-mass spectrometry (GC-MS). Multivariate pattern recognition and pathway analysis were used to analyze and interpret the data. Investigation of endogenous metabolic profiles revealed remarkable discrimination in both saliva and urine samples of the exposed population strongly suggesting the changes in metabolic composition within the identified metabolites (for urine samples: accuracy 0.9766, R2 = 0.9130, Q2 = 0.8703; for saliva samples, an accuracy of 0.9961, R2 = 0.9698, Q2 = 0.9637). Thirteen metabolites of urine samples and sixteen metabolites of saliva samples were identified as differential metabolites specific to pesticide exposure. Pathway analysis of differential metabolites revealed that amino acid metabolism, energy metabolism (glycolysis and TCA cycle) and glutathione metabolism (oxidative stress) were found to affect in pesticide exposed population. The present study suggested that GC-MS based metabolomics can help to reveal the metabolic perturbations in human population after pesticides exposure.

Metabolic oscillations on the circadian time scale in Drosophila cells lacking clock genes.

Circadian rhythms are cell-autonomous biological oscillations with a period of about 24 h. Current models propose that transcriptional feedback loops are the primary mechanism for the generation of circadian oscillations. Within this framework, Drosophila S2 cells are regarded as "non-rhythmic" cells, as they do not express several canonical circadian components. Using an unbiased multi-omics approach, we made the surprising discovery that Drosophila S2 cells do in fact display widespread daily rhythms. Transcriptomics and proteomics analyses revealed that hundreds of genes and their products, and in particular metabolic enzymes, are rhythmically expressed in a 24-h cycle. Metabolomics analyses extended these findings and demonstrate that central carbon metabolism and amino acid metabolism are core metabolic pathways driven by protein rhythms. We thus demonstrate that 24-h metabolic oscillations, coupled to gene and protein cycles, take place in nucleated cells without the contribution of any known circadian regulators. These results therefore suggest a reconsideration of existing models of the clockwork in Drosophila and other eukaryotic systems.

Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation.

A polyaromatic hydrocarbon degrading bacterium was isolated from a petroleum contaminated site and designated as Stenotrophomonas sp. strain IITR87. It was found to utilize pyrene, phenanthrene and benzo(a)pyrene as sole carbon source, but not anthracene, chrysene and fluoranthene. Gas chromatography and mass spectroscopy analysis resulted in identification of pyrene metabolites namely monohydroxypyrene, 4-oxa-pyrene-5-one, dimethoxypyrene and monohydroxyphenanthrene. Southern hybridization using naphthalene dioxygenase gene (nidA) as probe against the DNA of strain IITR87 revealed the presence of nidA gene. PCR analysis suggests dispersed occurrence of nid genes in the genome instead of a cluster as reported in a PAH-degrading Mycobacterium vanbaalenii PYR-1. The nid genes in strain IITR87, dioxygenase large subunit (nidA), naphthalene dioxygenase small subunit (nidB) and aldehyde dehydrogenase gene (nidD) showed more than 97 % identity to the reported nid genes from Mycobacterium vanbaalenii PYR-1. Most significantly, the biodegradation of PAHs was enhanced 25-60 % in the presence of surfactants rhamnolipid and Triton X-100 due to increased solubilization and bioavailability. These results could be useful for the improved biodegradation of high-molecular-weight PAHs in contaminated habitats.

Identifying the metabolic perturbations in earthworm induced by cypermethrin using gas chromatography-mass spectrometry based metabolomics.

Globally, cypermethrin is one of the most widely used synthetic pyrethroid for agricultural and domestic purposes. Most part of the pesticides used in the agriculture ends up as residues in the soil, making soil dwelling organisms, especially earthworms more susceptible to pesticide intoxication. Cypermethrin is known to be a neurotoxicant to many model organisms, including mammals and insects, but such type of toxicity evidence is not available for invertebrate systems like earthworms. In the present work, metabolomics based approach was utilized to identify the toxic mechanism of action of cypermethrin on earthworm (Metaphire posthuma) and these were exposed to sub-lethal concentrations of cypermethrin such as 2.5 mg/kg, 5 mg/kg, 10 mg/kg and 20 mg/kg (1/40(th), 1/20(th), 1/10(th) and 1/5(th) of LC50, respectively) for fourteen days. The results revealed that 22 metabolites (mainly fatty acids, sugars and amino acids) were shown significant responses in the exposed earthworms and these responses are dose dependent. It is proposed that mainly carbohydrate and fatty acids in neural system metabolism was disturbed. Overall, the results provided that metabolomics can be an effective tool to understand the effects of cypermethrin on the metabolic responses of earthworm Metaphire posthuma.

Activity-guided chemo toxic profiling of Cassia occidentalis (CO) seeds: detection of toxic compounds in body fluids of CO-exposed patients and experimental rats.

Our prior studies have shown an association between the deaths of children and consumption of Cassia occidentalis (CO) seeds. However, the chemicals responsible for the CO poisoning are not known. Therefore, the present study was designed to identify the key moieties in CO seeds and their cytotoxicity in rat primary hepatocytes and HepG2 cells. Activity-guided sequential extraction and fractionation of the seeds followed by GC-MS analysis identified the toxic compounds in the CO seeds. These identified compounds were subsequently detected and quantified in blood and urine samples from CO-exposed rats and CO poisoning human study cases. GC-MS analysis of different fractions of methanol extracts of CO seeds revealed the presence of five anthraquinones (AQs), viz. physcion, emodin, rhein, aloe-emodin, and chrysophanol. Interestingly, these AQs were detected in serum and urine samples from the study cases and CO-exposed rats. Cytotoxicity analysis of the above AQs in rat primary hepatocytes and HepG2 cells revealed that rhein is the most toxic moiety, followed by emodin, aloe-emodin, physcion, and chrysophanol. These studies indicate that AQ aglycones are responsible for producing toxicity, which may be associated with symptoms of hepatomyoencephalopathy in CO poisoning cases.

Determination of Urinary PAH Metabolites Using DLLME Hyphenated to Injector Port Silylation and GC-MS-MS.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and well-known carcinogens. Hydroxy derivatives of PAH are considered as biomarkers of PAH exposure, and there is a need to measure these metabolites at low concentrations. So, a precise and eco-friendly analytical method has been developed for rapid determination of PAH metabolites. For the first time, a new analytical method based on coupling of dispersive liquid-liquid microextraction (DLLME) with auto-injector port silylation (auto-IPS) followed by gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis is reported for the analysis of seven urinary PAH metabolites. Factors affecting DLLME and IPS, such as type and volume of extraction and disperser solvent, pH, ionic strength, injector port temperature, volume of N,O-bis(trimethylsilyl)trifluoroacetamide and type of solvent were investigated. Under optimized conditions, the limit of detection and limit of quantification were found to be in the range of 1-9 and 3-29 ng/mL, respectively. Satisfactory recoveries of metabolites in urine samples in the range of 87-95% were found. The developed method has been successfully applied for the determination of PAH metabolites in urine samples of exposed workers. DLLME-auto-IPS-GC-MS-MS method is time, labor, solvent and reagent saving, which can be routinely used for the analysis of urinary PAH metabolites.

Superoxide mediated photomodification and DNA damage induced apoptosis by Benz(a)anthracene via mitochondrial mediated pathway.

Benz(a)anthracene (BA) is an ubiquitous environmental pollutant of polycyclic aromatic hydrocarbon's (PAHs) family. We showed superoxide (O2(-)) catalyzed BA photo modification and apoptosis in HaCaT keratinocytes under sunlight exposure. O2(-) generation was confirmed by quenching through superoxide dismutase (SOD). BA induced photocytotoxicity were investigated through MTT and NRU assay. We proposed DNA insults such as single and double strand breakage and CPDs formation which results in cell cycle arrest and apoptosis by photosensitized BA. BA induced apoptosis was caspase dependent and occurred through a mitochondrial pathway. Reduction of mitochondrial membrane potential, translocation of Bax to mitochondria and cytochrome c release favors involvement of mitochondria in BA phototoxicity. AO/EB double staining and TEM analysis also support apoptotic cell death. We propose a p21 regulated apoptosis via expression of Bax, and cleaved PARP under sunlight exposure. Thus, we conclude that it is imperative to avoid solar radiation during peak hr (between 11A.M. and 3P.M.) when the amount of solar radiation is high, in the light of DNA damage which may lead to mutation or skin cancer through photosensitized BA under sunlight exposure. Concomitantly, investigation is urgently required for the photosafety of BA photoproducts reaching in the environment through photomodification.

Gas chromatography-mass spectrometry based metabolomic approach for optimization and toxicity evaluation of earthworm sub-lethal responses to carbofuran.

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.

Ultrasound assisted dispersive liquid-liquid microextraction followed by injector port silylation: a novel method for rapid determination of quinine in urine by GC-MS.

BACKGROUND: Silylation is a widely used derivatization method for the analysis of polar analytes by GC-MS. Ultrasound-assisted dispersive liquid-liquid microextraction (UA-DLLME) is an ecofriendly, rapid and simple microextraction method. For the first time, a novel approach has been developed and applied for the analysis of quinine in urine by combining UA-DLLME with injection port silylation. RESULTS: The LOD and LOQ were found to be 5.4 and 18 ng/ml. The intra- and inter-day precisions were less than 5 and 8%, respectively. Mean recoveries of quinine were found to be in the range of 87 to 96%. CONCLUSION: Ultrasound-assisted dispersive liquid-liquid microextraction is rapid, simple and consumes less reagent for the analysis of polar analytes such as quinine.