Potential new method of mixture effects testing using metabolomics and Caenorhabditis elegans.

The development of superior tools for molecular and computational biology in recent years has provided an opportunity for the creation of faster toxicological screens that are relevant for, but do not rely on, mammalian systems. In this study, NMR spectroscopy and GC-MS based metabolomics have been used in conjunction with multivariate statistics to examine the metabolic changes in the nematode Caenorhabditis elegans following exposure to different concentrations of the heavy metal nickel, the pesticide chlorpyrifos, and their mixture. Novel metabolic profiles were associated with both exposure and dose level. The biochemical responses were more closely matched when exposure was at the same effect level, even for different chemicals, than when exposure was for different levels of the same chemical (e.g., low versus high dose). Responses to the mixture reflected the contribution of the chemicals to the overall exposure. In common with the metabolic responses of several other species exposed to the same chemicals, we observed changes in branch chain amino acids and tricarboxylic acid cycle intermediates. These results form the basis for a rapid and economically viable toxicity test that defines the molecular effects of pollution/toxicant exposure in a manner that is relevant to higher vertebrates.

A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus rubellus.

A major challenge in ecotoxicology is to understand the effects of multiple toxicants on organisms. Here we assess the effects on survival, weight change, cocoon production and metabolism caused by exposure to two similarly acting (imidacloprid/thiacloprid) and two dissimilarly acting (chlorpyrifos/Nickel) chemicals on the earthworm Lumbricus rubellus. We assessed the standard models of concentration addition (CA) and independent action (IA), in conjunction with a metabolomics based approach to elucidate mechanisms of effect. For imidacloprid and thiacloprid the reproductive effects indicated probable additivity. Although this suggests joint effects through a similar mechanism, metabolite changes for each pesticide actually indicated distinct effects. Further, earthworms exposed to a 0.5 toxic unit equitoxic mixture demonstrated metabolic effects intermediate between those for each pesticide, indicating a non-interactive, independent joint effect. For higher effect level mixtures (1 and 1.5 toxic units), metabolite changes associated with thiacloprid exposure began to dominate. The metabolomic effects of the two dissimilarly acting chemicals were distinct, confirming separate modes of action and both proved more toxic than anticipated from previous studies. In the mixtures, phenotypic effects were in accordance with IA estimates, while metabolite changes were dominated by Ni effects, even though chlorpyrifos contributed most to reproductive toxicity. This could be attributed to the greater systematic effect of Ni when compared to the more specifically acting chlorpyrifos.

Therapeutic Efficacy of Difluprednate 0.05% Versus Prednisolone Acetate 1% in Controlling Inflammation and Macular Oedema Following Phacoemulsification: An Optical Coherence Tomography-Based Study.

BACKGROUND: Topical corticosteroids have been the cornerstone in the management of postoperative inflammation following cataract surgery. Due to potential side effects of the older topical steroids like prednisolone acetate and dexamethasone or betamethasone, newer potent steroids preparation like difluprednate, loteprednol or fluorometholone are now being used at lesser dose and frequency to control inflammation. There is scanty literature on the efficacy of these drugs in preventing inflammation and macular oedema in the Indian population. PURPOSE: The purpose of this study was to compare the efficacy of difluprednate 0.05% against prednisolone 1% eye drops for control of inflammation following phacoemulsification. The adverse effects of both drugs were also evaluated in this retrospective study. METHODS: This retrospective cohort study included 181 patients operated for age-related cataracts by a single surgeon at a tertiary referral eye hospital between December 2018 and March 2019. Patients received either difluprednate 0.05% emulsion (n=90 eyes) or prednisolone acetate 1% (n=91 eyes) after phacoemulsification with the same brand intraocular lens (IOL) and same phaco machine. The topical medication was initiated one day postoperatively and continued for six weeks in tapering dosage. Pain score (Visual Analogue Scale, VAS), conjunctival hyperemia, anterior chamber (AC) cell grading, corneal oedema, central retinal thickness, subclinical cystoid macular oedema (SCME), intraocular pressures (IOP) and best-corrected visual acuity (BCVA) were examined after one week and six weeks of cataract extraction.  Results: There was no statistically significant difference observed with regards to pain score (no pain in any patients after six weeks), conjunctival hyperemia (no hyperemia in any patients after six weeks), AC inflammation (no reaction in any patients), central retinal thickness (234.44 ± 35.75µ vs. 234.8 ± 34.99µ, p-value 0.946), SCME (16.67% vs. 13.19%, p-value 0.511), IOP (16.8 vs. 15.47 mmHg, p-value 0.101) and BCVA (BCVA 6/6 in 57.7% vs. 70.32%, p-value >0.05) between both groups. The mean change in IOP in both the groups at one week (0 ± 4.4 vs. 1.87 ± 3.54, p-value 0.0007) and six weeks (-0.01 ± 5.53 vs. 1.88 ± 4.01, p-value 0.004) was significant.  Conclusion: Both the groups were equivalent with regards to their therapeutic efficacy in controlling postoperative inflammation and restoration of vision following phacoemulsification.

Spectrum of Ocular Findings in Closed Head Injuries, Correlation With Severity of Neurological Involvement, and Treatment Outcome: A Hospital-Based Cross-Sectional Study.

PURPOSE: To study the various ocular findings in patients with closed head injuries, to find any association with the degree of neurological involvement, and to analyze the treatment outcome after the necessary intervention. SETTING: Tertiary referral hospital in Eastern India. DESIGN:  Prospective observational study. METHODS: Patients with closed head injuries attending our Outpatient department as well as referred from the Neurosurgery department for ophthalmic evaluation between October 2017 and September 2019 were recruited for the study. All patients meeting the inclusion criteria were examined by an experienced ophthalmologist. The Glasgow coma scale (GCS) was applied to grade the neurological involvement by the neurosurgery team. Ocular findings were recorded and necessary imaging was requested. Appropriate neurosurgery consultations were done in patients with neurological findings. All ocular injuries were managed as per institutional protocol. Descriptive statistics were used for analysis with p< 0.05 taken as statistically significant. RESULTS: A total of 207 patients (414 eyes) were included in the study. The mean age was 33.82 years, with the prevalence of male patients (82.12%). The most common cause of head injury was RTA (57.01%) followed by assault (11.59%). The majority of patients (53.14%) were classified as having moderate, 46.37% patients with mild, and 0.48% with severe neurological involvement as per GCS scoring. Isolated ocular findings were seen in 70.04% of patients while 29.95% of patients had both neurological and ophthalmic features. Ocular adnexal involvement was observed in 38.6%, anterior segment involvement in 86%, neuro-ophthalmic manifestations in 33.3%, and posterior segment involvement in 38.6% of patients. Ocular signs were resolved over due course of time in 48.8% of patients, completely resolved in 28%, while there was no improvement in 6.28% of patients. The final best-corrected visual acuity of >6/18 was achieved in 51.69% of patients. Statistical significance was observed in the correlation between the GCS scoring and general ocular findings (p= 0.02) as well as a relative afferent pupillary defect (p=0.003). The association between age > 50 years and neuro-ophthalmic features was not found to be statistically significant (p=0.56). CONCLUSION: Poor visual acuity at presentation, optic canal fractures, the presence of multiple fractures of orbital walls, no improvement in vision within 48 hours of starting intravenous corticosteroids, were indicators of a poor visual prognosis in this study. The GCS, neuro-deficit, and ocular signs contribute significantly to the prediction of outcomes. Prompt treatment and referral can lead to a good resolution of symptoms and signs.

Gene expression profiling to characterize sediment toxicity--a pilot study using Caenorhabditis elegans whole genome microarrays.

BACKGROUND: Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. RESULTS: In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. CONCLUSION: This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments.

Hydrogen sulfide is an endogenous regulator of aging in Caenorhabditis elegans.

AIMS: To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS: We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine ß synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas. INNOVATION: The possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study. CONCLUSION: Our results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

Transcript expression patterns illuminate the mechanistic background of hormesis in caenorhabditis elegans maupas.

The animal model Caenorhabditis elegans was employed to study polyphenol- and humic substances-induced hormetic changes in lifespan. A detailed insight into the underlying mechanism of hormesis was uncovered by applying whole genome DNA microarray experimentation over a range of quercetin (Q), tannic acid (TA), and humic substances (HuminFeed(®), HF) concentrations. The transcriptional response to all exposures followed a non-linear mode which highlighted differential signaling and metabolic pathways. While low Q concentrations regulated processes improving the health of the nematodes, higher concentrations extended lifespan and modulated substantially the global transcriptional response. Over-represented transcripts were notably part of the biotransformation process: enhanced catabolism of toxic intermediates possibly contributes to the lifespan extension. The regulation of transcription, Dauer entry, and nucleosome suggests the presence of distinct exposure dependent differences in transcription and signaling pathways. TA- and HF-mediated transcript expression patterns were overall similar to each other, but changed across the concentration range indicating that their transcriptional dynamics are complex and cannot be attributed to a simple adaptive response. In contrast, Q-mediated hormesis was well aligned to fit the definition of an adaptive response. Simple molecules are more likely to induce an adaptive response than more complex molecules.

The Nematode Caenorhabditis elegans, Stress and Aging: Identifying the Complex Interplay of Genetic Pathways Following the Treatment with Humic Substances.

Low concentrations of the dissolved leonardite humic acid HuminFeed(®) (HF) prolonged the lifespan and enhanced the thermal stress resistance of the model organism Caenorhabditis elegans. However, growth was impaired and reproduction delayed, effects which have also been identified in response to other polyphenolic monomers, including Tannic acid, Rosmarinic acid, and Caffeic acid. Moreover, a chemical modification of HF, which increases its phenolic/quinonoid moieties, magnified the biological impact on C. elegans. To gain a deep insight into the molecular basis of these effects, we performed global transcriptomics on young adult (3 days) and old adult (11 days) nematodes exposed to two different concentrations of HF. We also studied several C. elegans mutant strains in respect to HF derived longevity and compared all results with data obtained for the chemically modified HF. The gene expression pattern of young HF-treated nematodes displayed a significant overlap to other conditions known to provoke longevity, including various plant polyphenol monomers. Besides the regulation of parts of the metabolism, transforming growth factor-beta signaling, and Insulin-like signaling, lysosomal activities seem to contribute most to HF's and modified HF's lifespan prolonging action. These results support the notion that the phenolic/quinonoid moieties of humic substances are major building blocks that drive the physiological effects observed in C. elegans.

Meta-Analysis of Global Transcriptomics Suggests that Conserved Genetic Pathways are Responsible for Quercetin and Tannic Acid Mediated Longevity in C. elegans.

Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of Caenorhabditis elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to three concentrations of Quercetin or Tannic acid, respectively. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published datasets derived from (i) longevity mutants or (ii) infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the prediction of putative mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling, and the p38 MAPK pathway and Tannic acid's impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, and genetic players of the p38 MAPK pathway therefore seem to be crucial regulators for both polyphenols. Taken together, this study underlines how meta-analyses can provide an insight of molecular events that go beyond the traditional categorization into gene ontology-terms and Kyoto encyclopedia of genes and genomes-pathways. It also supports the call to expand the generation of comparative and integrative databases, an effort that is currently still in its infancy.

Bio-electrospraying the nematode Caenorhabditis elegans: studying whole-genome transcriptional responses and key life cycle parameters.

Bio-electrospray, the direct jet-based cell handling approach, is able to handle a wide range of cells (spanning immortalized, primary to stem cells). Studies at the genomic, genetic and the physiological levels have shown that, post-treatment, cellular integrity is unperturbed and a high percentage (more than 70%, compared with control) of cells remain viable. Although, these results are impressive, it may be argued that cell-based systems are oversimplistic. Therefore, it is important to evaluate the bio-electrospray technology using sensitive and dynamically developing multi-cellular organisms that share, at least some, similarities with multi-cell microenvironments encountered with tissues and organs. This study addressed this issue by using a well-characterized model organism, the non-parasitic nematode Caenorhabditis elegans. Nematode cultures were subjected to bio-electrospraying and compared with positive (heat shock) and negative controls (appropriate laboratory culture controls). Overall, bio-electrospraying did not modulate the reproductive output or induce significant changes in in vivo stress-responsive biomarkers (heat shock proteins). Likewise, whole-genome transcriptomics could not identify any biological processes, cellular components or molecular functions (gene ontology terms) that were significantly enriched in response to bio-electrospraying. This demonstrates that bio-electrosprays can be safely applied directly to nematodes and underlines its potential future use in the creation of multi-cellular environments within clinical applications.