Performance of Metagenomic Next-Generation Sequencing of Cell-Free DNA From Vitreous and Aqueous Humor for Diagnoses of Intraocular Infections.

BACKGROUND: Delayed diagnosis and improper therapy for intraocular infections usually result in poor prognosis. Due to limitations of conventional culture and polymerase chain reaction methods, most causative pathogens cannot be identified from vitreous humor (VH) or aqueous humor (AH) samples with limited volume. METHODS: Patients with suspected intraocular infections were enrolled from January 2019 to August 2021. Metagenomic next-generation sequencing (mNGS) was used to detected causative pathogens. RESULTS: This multicenter prospective study enrolled 488 patients, from whom VH (152) and AH (336) samples were respectively collected and analyzed using mNGS of cell-free DNA (cfDNA). Taking final comprehensive clinical diagnosis as the gold standard, there were 39 patients with indefinite final diagnoses, whereas 288 and 161 patients were diagnosed as definite infectious and noninfectious diseases, respectively. Based on clinical adjudication, the sensitivity (92.2%) and total coincidence rate (81.3%) of mNGS using VH samples were slightly higher than those of mNGS using AH samples (85.4% and 75.4%, respectively). CONCLUSIONS: Using mNGS of cfDNA, an era with clinical experience for more rapid, independent, and impartial diagnosis of bacterial and other intraocular infections can be expected.

Ascorbic acid export from human donor lenses: Is the lens a source of ascorbic acid in the ocular humors?

In previous work, we have shown that the lens acts a reservoir of the antioxidant glutathione (GSH), capable of exporting this antioxidant into the ocular humors and potentially protecting the tissues of the eye that interface with these humors from oxidative stress. In this study, we have extended this work by examining whether the lens acts as a source of ascorbic acid (AsA) to maintain the high levels of AsA known to be present in the ocular humors either by the direct export of AsA into the humors and/or by functioning as a recycling site for AsA, via the direct uptake of oxidised ascorbate (DHA) from the humors, its regeneration to AsA in the lens and then its subsequent export back into the humors. To test this, human lenses of varying ages were cultured for 1 h under hypoxic conditions and AsA/DHA levels measured in the media and in the lens. Human lenses were also cultured in compartmentalised chambers to determine whether efflux of AsA/DHA occurs at the anterior or posterior surface. Immunohistochemistry was performed on human donor lenses and sections labelled with antibodies against GLUT1, a putative DHA uptake transporter. Vitreous humor was collected from patients undergoing vitrectomy who either had a natural clear lens, an artificial intraocular implant (IOL) or a cataractous lens, and AsA/DHA and GSH and oxidised GSH (GSSG) measured. We found that cultured human donor lenses released both AsA and DHA into the media. Culturing of lenses in a compartmentalised chamber revealed that AsA and DHA efflux occurs at both surfaces, with relatively equal amounts of AsA and DHA released from each surface. The posterior surface of the lens was shown to express the GLUT1 transporter. Analysis of vitreous samples from patients undergoing vitrectomy revealed that vitreous GSH and AsA levels were similar between the natural lens group, IOL and cataractous lens group. Taken together, while human donor lenses were shown to export AsA and DHA into the surrounding media, the amount of AsA and DHA released from donor lenses was low and not sufficient to sustain the high levels of total AsA normally present in the humors. This suggests that although the lens is not the main source for maintaining high levels of AsA in the ocular humors, the lens may help to support local AsA levels close to the lens.

Exosomal lncRNA-MIAT promotes neovascularization via the miR-133a-3p/MMP-X1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.

CircRNA expression profiles and regulatory networks in the vitreous humor of people with high myopia.

Myopia is a global health and economic issue. Circular RNAs (circRNAs) have been shown to play an important role in the pathogenesis of many ocular diseases. We first evaluated the circRNA profiles and possible roles in vitreous humor samples of individuals with high myopia by a competitive endogenous RNA (ceRNA) array. Vitreous humor samples were collected from 15 high myopic (5 for ceRNA array, and 10 for qPCR) and 15 control eyes (5 for ceRNA array, and 10 for qPCR) with idiopathic epiretinal membrane (ERM) and macular hole (MH). 486 circRNAs (339 upregulated and 147 downregulated) and 264 mRNAs (202 upregulated and 62 downregulated) were differentially expressed between the high myopia and control groups. The expression of hsa_circ_0033079 (hsa-circDicer1), hsa_circ_0029989 (hsa-circNbea), hsa_circ_0019072 (hsa-circPank1) and hsa_circ_0089716 (hsa-circEhmt1) were validated by qPCR. Pearson analysis and multivariate regression analysis showed positive and significant correlations for axial length with hsa-circNbea and hsa-circPank1. KEGG analysis showed that the target genes of circRNAs were enriched in the mTOR, insulin, cAMP, and VEGF signaling pathways. GO analysis indicated that circRNAs mainly targeted transcription, cytoplasm, and protein binding. CircRNA-associated ceRNA network analysis and PPI network analysis identified several critical genes for myopia. The expression of circNbea, circPank1, miR-145-5p, miR-204-5p, Nras, Itpr1 were validated by qPCR in the sclera of form-deprivation myopia (FDM) mice model. CircPank1/miR-145-5p/NRAS and circNbea/miR-204-5p/ITPR1 were identified and may be important in the progression of myopia. Our findings suggest that circRNAs may contribute to the pathogenesis of myopia and may serve as potential biomarkers.

Age-related changes on physicochemical properties of the artificial vitreous humor: A practical tool for enhancing ex vivo studies.

The vitreous humor (VH) is a hydrophilic, jelly-like ocular fluid, which is located in the posterior chamber of the eye. The rheological, structural, and chemical properties of VH change significantly during aging, which further causes eye-associated diseases and could be a potential indicator for various diseases. In this study, artificial VH (A-VH) samples were created by taking into account different age groups to observe age-related changes in the physicochemical properties of these samples. This study aimed to measure the physicochemical properties of age-dependently prepared A-VH samples to determine the changes with aging in the physicochemical properties of A-VH samples. Phosphate-buffered saline (PBS)-based A-VH samples were prepared in three types representing adult, middle-aged, and elder individuals. Age-related changes in physicochemical properties (surface tension, osmolality, pH, relative viscosity, density, and refractive index) were analyzed by related equipment. The A-VH samples, prepared using PBS, showed strong similarity to authentic VH in terms of physicochemical properties. While the age-related changes studies have revealed some discrepancies between age-dependently prepared A-VH samples in terms of surface tension, osmolality, relative viscosity, and pH with high correlation coefficients (r2 > 0,94), density and refractive index values did not show any significant differences and correlation between types of A-VH representing 3 age groups. In conclusion, age-dependent A-VH samples were created successfully to use ex vivo method development studies, and the influence of aging on the physicochemical properties of VH was demonstrated as well.

Extracellular vesicle biomarkers in ocular fluids associated with ophthalmic diseases.

Extracellular vesicles (EVs) are released as highly stable lipid bilayer particles carrying proteins, lipids, glycans and miRNAs. The contents of EVs vary based on the cellular origin, biogenesis route and the functional state of the cell suggesting certain diseased conditions. A growing body of evidence show that EVs carry important molecules implicated in the development and progression of ophthalmic diseases. EVs associated with ophthalmic diseases are mainly carried by one of the three ocular biofluids which include tears, aqueous humor and vitreous humor. This review summarizes the list of EV derived biomarkers identified thus far in ocular fluids for ophthalmic disease diagnosis. Further, the methods used for sample collection, sample volume and the sample numbers used in these studies have been highlighted. Emphasis has been given to describe the EV isolation and the characterization methods used, EV size profiled and the EV concentrations analyzed by these studies, thus providing a roadmap for future EV biomarker studies in ocular fluids.

Silk fibroin/vitreous humor hydrogel scaffold modified by a carbodiimide crosslinker for wound healing.

Natural-derived biomaterials can be used as substrates for the growth, proliferation, and differentiation of cells. In this study, bovine vitreous humor as a biological material was cross-linked to silk fibroin with different concentration ratios to design a suitable substrate for corneal tissue regeneration. The cross-linked samples were evaluated with different analyses such as structural, physical (optical, swelling, and degradation), mechanical, and biological (viability, cell adhesion) assays. The results showed that all samples had excellent transparency, especially those with higher silk fibroin content. Increasing the ratio of vitreous humor to silk fibroin decreased mechanical strength and increased swelling and degradation, respectively. There was no significant difference in the toxicity of the samples, and with the increase in vitreous humor ratio, adhesion and cell proliferation increased. Generally, silk fibroin with vitreous humor can provide desirable characteristics as a transparent film for corneal wound healing.

Applications of artificial intelligence and bioinformatics methodologies in the analysis of ocular biofluid markers: a scoping review.

PURPOSE: This scoping review summarizes the applications of artificial intelligence (AI) and bioinformatics methodologies in analysis of ocular biofluid markers. The secondary objective was to explore supervised and unsupervised AI techniques and their predictive accuracies. We also evaluate the integration of bioinformatics with AI tools. METHODS: This scoping review was conducted across five electronic databases including EMBASE, Medline, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Web of Science from inception to July 14, 2021. Studies pertaining to biofluid marker analysis using AI or bioinformatics were included. RESULTS: A total of 10,262 articles were retrieved from all databases and 177 studies met the inclusion criteria. The most commonly studied ocular diseases were diabetic eye diseases, with 50 papers (28%), while glaucoma was explored in 25 studies (14%), age-related macular degeneration in 20 (11%), dry eye disease in 10 (6%), and uveitis in 9 (5%). Supervised learning was used in 91 papers (51%), unsupervised AI in 83 (46%), and bioinformatics in 85 (48%). Ninety-eight papers (55%) used more than one class of AI (e.g. > 1 of supervised, unsupervised, bioinformatics, or statistical techniques), while 79 (45%) used only one. Supervised learning techniques were often used to predict disease status or prognosis, and demonstrated strong accuracy. Unsupervised AI algorithms were used to bolster the accuracy of other algorithms, identify molecularly distinct subgroups, or cluster cases into distinct subgroups that are useful for prediction of the disease course. Finally, bioinformatic tools were used to translate complex biomarker profiles or findings into interpretable data. CONCLUSION: AI analysis of biofluid markers displayed diagnostic accuracy, provided insight into mechanisms of molecular etiologies, and had the ability to provide individualized targeted therapeutic treatment for patients. Given the progression of AI towards use in both research and the clinic, ophthalmologists should be broadly aware of the commonly used algorithms and their applications. Future research may be aimed at validating algorithms and integrating them in clinical practice.

Polymer coated polymeric microneedles for intravitreal delivery of dexamethasone.

The polymer coated polymeric (PCP) microneedles (MNs) is a novel approach for controlled delivery of drugs (without allowing release of the excipients) to the target site. PCP MNs was explored as an approach to deliver the drug intravitreally to minimize the risks associated with conventional intravitreal injections. The core MNs was fabricated with polyvinyl pyrrolidone K30 (PVP K30) and coating was with Eudragit E100. Preformulation studies revealed that the films prepared using Eudragit E 100 exhibited excellent integrity in the physiological medium after prolonged exposure. FTIR studies were performed to investigate the possible interaction between the API and the polymer. The PCP MNs fabricated with different drug loads (dexamethasone sodium phosphate) were subjected to in vitro drug release studies. The drug release from uncoated MNs was instantaneous and complete. On the other hand, a controlled release profile was observed in case of PCP MNs. Likewise, even in the ex vivo porcine eye model, the drug release was gradual into the vitreous humor in case of PCP MNs. The uncoated microneedles released all the drug instantaneously where the PCP MNs retarded the release up to 3 h.

Detection of microRNAs expression signatures in vitreous humor of intraocular tuberculosis.

BACKGROUND: MicroRNA (miRNA) expression analysis has been shown to provide them as biomarkers in several eye diseases and has a regulatory role in pathogenesis. However, miRNA expression analysis in the vitreous humor (VH) of intraocular tuberculosis (IOTB) is not studied. Thus, we aim to find miRNA expression signatures in the VH of IOTB patients to identify their regulatory role in disease pathogenesis and to find them as potential biomarkers for IOTB. METHODS AND RESULTS: First, we profiled miRNAs in VH of three IOTB and three Macular hole (MH) samples as controls through small-RNA deep sequencing using Illumina Platform. In-house bioinformatics analysis identified 81 dysregulated miRNAs in IOTB. Further validation in VH of IOTB (n = 15) compared to MH (n = 15) using Real-Time quantitative PCR (RT-qPCR) identified three significantly upregulated miRNAs, hsa-miR-150-5p, hsa-miR-26b-5p, and hsa-miR-21-5p. Based on the miRNA target prediction, functional network analysis, and RT-qPCR analysis of target genes, the three miRNAs downregulating WNT5A, PRKCA, MAP3K7, IL7, TGFB2, IL1A, PRKCB, TNFA, and TP53 genes involving MAPK signaling pathway, PI3K-AKT signaling pathway, WNT signaling pathway, Cell cycle, TGF-beta signaling pathway, Long-term potentiation, and Sphingolipid signaling pathways, have a potential role in disease pathogenesis. The ROC analysis of RT-qPCR data showed that hsa-miR-150-5p with AUC = 0.715, hsa-miR-21-5p with AUC = 0.789, and hsa-miR-26b-5p with AUC = 0.738; however, the combination of hsa-miR-21-5p and hsa-miR-26b-5p with AUC = 0.796 could serve as a potential biomarker for IOTB. CONCLUSIONS: This study provides the first report on miRNA expression signatures detected in VH for IOTB pathogenesis and also provides a potential biomarker for IOTB.

High risk and low prevalence diseases: Endophthalmitis.

INTRODUCTION: Endophthalmitis is a serious, vision-threatening condition that carries with it a high rate of morbidity. OBJECTIVE: This review highlights the pearls and pitfalls of endophthalmitis, including presentation, diagnosis, and management in the emergency department (ED) based on current evidence. DISCUSSION: Endophthalmitis is a vision-threatening emergency associated with infection and inflammation of vitreous and aqueous humor. Risk factors include ocular trauma or surgery, immunocompromised state, diabetes mellitus, and injection drug use. History and examination include visual changes, ocular pain, and inflammatory findings (e.g., hypopyon). Fever may be present. Diagnosis should be based on the clinical evaluation, though aqueous or vitreous culture performed by the ophthalmology specialist is recommended. Imaging including computed tomography, magnetic resonance imaging, and ultrasound may suggest the disease but cannot exclude the diagnosis. Management includes emergent ophthalmology consultation and evaluation. Treatment for all types of endophthalmitis is injection of intravitreal antibiotics with consideration of vitrectomy in severe cases. Systemic antimicrobials are recommended in specific types of endophthalmitis. Prompt recognition and diagnosis are key to optimizing favorable visual outcomes. CONCLUSIONS: An understanding of endophthalmitis can assist emergency clinicians in diagnosing and managing this serious disease.

IPMICALC: an Integrated Post-mortem Interval Calculator.

Correctly estimating the post-mortem interval (PMI) is essential in forensic practice. In recent decades, the measurement of vitreous humor analyte concentrations - especially of hypoxanthine and potassium - and their correlation with the PMI have returned good results. However, calculating the PMI from the data collected can be quite complex. The present paper describes a web resource ( http://modestya.usc.es:3838/Forensic/ ) that simplifies the procedure. The PMI is determined (with its 95% confidence interval) in a rapid, easy, and reliable manner based on the use of mathematical models, the biochemistry of the vitreous humor, and physical variables such as the ambient temperature, the rectal temperature, and bodyweight. The application is entirely free to use.

Serum and vitreous vascular endothelial growth factor levels in diabetic retinopathy.

PURPOSE: To research whether serum vascular endothelial growth factor (VEGF) levels could be used to evaluate diabetic retinopathy (DR) progression and to compare vitreous VEGF levels after injections of intravitreal bevacizumab (IVB), ranibizumab (IVR), and triamcinolone acetonide (IVTA) in proliferative diabetic retinopathy (PDR). METHODS: We enrolled a total of 91 eyes of 89 subjects (70 eyes of 68 diabetics and 21 eyes of 21 non-diabetic controls). The diabetic subjects were divided into three groups as PDR (n = 28), non-proliferative diabetic retinopathy (n = 20), and no-DR (n = 20). Eyes with PDR (n = 31) were injected with IVB (n = 7), IVR (n = 10), or IVTA (n = 6) 3 days before vitrectomy, and eight eyes did not receive an injection. Serum and vitreous samples were collected before vitrectomy and analyzed using ELISA. RESULTS: We found the severity of retinopathy was not correlated with serum VEGF levels (P = .919, ρ = -0.011). Compared with the controls, vitreous VEGF was higher in the PDR (P < .001), whereas serum VEGF did not differ (P = .99). The controls had lower vitreous VEGF than the IVB, IVR, and no-injection subgroups (P = .01, P < .001, and P = .04, respectively). Vitreous VEGF was similar among the injected and no-injection subgroups (P = .17). CONCLUSIONS: Serum VEGF levels may not directly reflect retinopathy progression. Neither IVB, IVR nor IVTA could eliminate vitreous VEGF levels within 3 days before vitrectomy.

Changes of 1, 5-AG in Vitreous Humor of Rabbit Cadavers with Hyperglycemic Metabolism.

OBJECTIVES: To investigate the concentration and change characteristics of 1, 5-anhydroglucitol (1, 5-AG) in the vitreous humor of rabbit cadavers with hyperglycemic metabolism, and to explore the value of 1, 5-AG in forensic pathology identification of death caused by hyperglycemic metabolism disorders. METHODS: A diabetic hyperglycemic rabbit model was established by using alloxan. Eighteen rabbits with fasting glucose concentration ≥13.80 mmol/L (experimental group) and 18 healthy rabbits with fasting glucose concentration ≤6.10 mmol/L (control group) were selected. After death from air embolism. The blood samples were collected immediately, and vitreous humor samples were collected at 0 h, 12 h, 24 h and 36 h after death. The concentration of 1, 5-AG in the blood and vitreous humor of rabbits was determined. RESULTS: The blood glucose concentration in the experimental group was (25.10±3.14) mmol/L. At the time of death, there was no significant difference in the concentration of 1, 5-AG in the blood [(0.94±0.20) µg/mL] and in the vitreous humor (0.99±0.05 µg/mL, P>0.05). The concentration of 1, 5-AG in the vitreous humor of the experimental group was lower than that of the corresponding control group at all time points (P<0.05), and there was no significant difference betwwen 1, 5-AG concentration in vitreous humor between earch time point in the experimental group and the control group (P>0.05). Correlation analysis showed that the concentration of 1,5-AG in blood was negatively correlated with blood glucose in both control group and experimental group (control group: r=-0.79, P<0.05; experimental group: r=-0.97, P<0.05). CONCLUSIONS: Vitreous humor can replace blood as an effective test sample for 1,5-AG detection. The concentration of 1, 5-AG in rabbit vitreous humor remains stable within 36 hours after death and is not affected by the change of postmortem interval. If the concentration of 1, 5-AG decreases significantly, it indicates the existence of hyperglycemia in rabbits before death.

Research Progress on Estimation of Postmortem Interval Based on Ocular Tissues Structure.

Estimation of postmortem interval (PMI) is one of the important research contents in forensic pathology, and it has always been the focus and hot spot of research work. In recent years, scholars at home and abroad have made some research progress in estimating PMI by using ocular tissue. After death, the changes of cornea, aqueous humor, iris, lens, vitreous humor and retina all show time sequence change rule highly related to PMI. This paper reviews the research progress of PMI estimation based on the morphological, biochemical, molecular and genetic material changes of different ocular tissue structures after death, and discusses the existing problems and development trends.

REM phase: An ingenious mechanism to enhance clearance of metabolic waste from the retina.

The Rapid Eye Movement (REM) phase of sleep, also known as "active sleep" because of physiological similarities to waking state, is characterized by intense cerebral electrical activity, propensity to dream vividly and suppression of skeletal muscle activity (atonia) except for the extraocular muscles which give rise to the so-called REM. In 1998 David Maurice, an ophthalmologist, proposed that REM sleep was associated with an eye function: it would be required to stir the anterior chamber and bath it with aqueous humor to prevent corneal anoxia during sleep. However, potential metabolic problems could arise in the outer retinal layers which lack a direct blood supply. New research lends support to the hypothesis that a para-vascular transport system, the so-called "glymphatic", is present in the eye analogous to the one recently discovered in the brain. It is a functional waste clearance pathway which promotes elimination of interstitial solutes from the brain along para-vascular channels. Glymphatic function increases during sleep and just as a "brain pump" moves fluids in the central nervous system, a "vitreous pump" moves them into the eyeballs during REM phase. A number of similarities between Alzheimer's disease and several retinal degenerations have been described, particularly with respect to either age-related macular degeneration and chronic open-angle glaucoma. Impairment of this mechanism in some disease states and in the normal aging process could have serious consequences for visual function. In this manuscript I propose a new hypothesis regarding the role of REM phase on physio-pathology of the human eye: it would be an ingenious mechanism to enhanced clearance of metabolic waste from the retina.

Use of cadaveric vitreous humor as an innovative substrate for diatoms research and forensic diagnosis of drowning.

The search for diatoms is the test that most of all may contribute to the forensic diagnosis of drowning. Diatoms can be extracted through different methods, which are all complex and long. In this preliminary study, we assessed human vitreous humor as an innovative substrate on which to research diatoms. Sampling and analyses were performed on 3 groups of 10 corpses each. The first one was composed of drowned victims. The second one of victims of traffic accidents with subsequent projection in water; based on the severe traumatic injuries reported at autopsy, we considered such victims as ideally assimilable to bodies immersed in water postmortem. The third group was composed of subjects who died for natural causes and without any relationship with water. The vitreous humor was centrifugated and cytocentrifuged and spotted on two different histological slides: one was left blank and the other one was assessed with hematoxylin and eosin staining. Microscopic observation successfully revealed diatoms in all cases of the first group; in the second group diatoms were not detected in 6 cases; in the third group, no diatoms at all were observed. Diatoms were always qualitatively and quantitatively consistent with those found in the corresponding viscera and waters, which had been investigated through classical acid digestion. Our analyses have demonstrated that the vitreous humor behaves in a completely similar way to the other viscera in cases of drowning. Although further investigations are necessary, vitreous humor has proved to be an innovative, suitable, and reliable substrate for the forensic research of diatoms.

A sustainable approach for the stability study of psychotropic substances using vitreous humor and liver as alternative matrices.

The stability of psychotropic substances representing various drug groups important from the perspective of forensic chemistry, including benzodiazepines, antidepressants, carbamazepine, cocaine, and their selected metabolites, was investigated for 1 month in two alternative biological matrices, vitreous humor and liver homogenate. Three different thermal storage conditions (-20, 4, and 20 °C) were tested. Liquid chromatography-mass spectrometry (LC-MS) analysis was preceded by an effective solid-phase microextraction (SPME) procedure. The results were statistically analyzed using one-way ANOVA to find significant concentration variations over time. The results obtained allowed for dividing the analytes into four groups: stable under all tested conditions, only at -20 and 4 °C, only at 20 °C, and overall unstable. Nordiazepam, venlafaxine, and cocaine and its metabolites turned out to be the most unstable substances, while fluoxetine showed the highest storage stability in both matrices. The SPME/LC-MS method was comprehensively evaluated according to the principles of white analytical chemistry (WAC), which reconcile the greenness and functionality of the method. A close to 100% whiteness score proves its sustainability and suitability for the intended application.

Inferring Postmortem Submersion Interval in Rats Found in Water Based on Vitreous Humor Metabolites.

OBJECTIVES: The metabolomics technique of LC-MS/MS combined with data analysis was used to detect changes and differences in metabolic profiles in the vitreous humor of early rat carcasses found in water, and to explore the feasibility of its use for early postmortem submersion interval (PMSI) estimation and the cause of death determination. METHODS: The experimental model was established in natural lake water with 100 SD rats were randomly divided into a drowning group (n=50) and a postmortem (CO2 suffocation) immediately submersion group (n=50). Vitreous humor was extracted from 10 rats in each group at 0, 6, 12, 18 and 24 h postmortem for metabolomics analyses, of which 8 were used as the training set to build the model, and 2 were used as test set. PCA and PLS multivariate statistical analysis were performed to explore the differences in metabolic profiles among PMSI and causes of death in the training set samples. Then random forest (RF) algorithm was used to screen several biomarkers to establish a model. RESULTS: PCA and PLS analysis showed that the metabolic profiles had time regularity, but no differences were found among different causes of death. Thirteen small molecule biomarkers with good temporal correlation were selected by RF algorithm. A simple PMSI estimation model was constructed based on this indicator set, and the data of the test samples showed the mean absolute error (MAE) of the model was 0.847 h. CONCLUSIONS: The 13 metabolic markers screened in the vitreous humor of rat corpses in water had good correlations with the early PMSI. The simplified PMSI estimation model constructed by RF can be used to estimate the PMSI. Additionally, the metabolic profiles of vitreous humor cannot be used for early identification of cause of death in water carcasses.

Preliminary Metabolomic Profiling of the Vitreous Humor from Hypothermia Fatalities.

The postmortem diagnosis of hypothermia fatalities is often complex due to the absence of pathognomonic lesions and biomarkers. In this study, potential novel biomarkers of hypothermia fatalities were searched in the vitreous humor of known cases of hypothermia fatalities (n = 20) compared to control cases (n = 16), using a targeted metabolomics approach allowing quantitative detection of 188 metabolites. A robust discriminant model with good predictivity was obtained with the supervised OPLS-DA multivariate analysis, showing a distinct separation between the hypothermia and control groups. This signature was characterized by the decreased concentrations of five metabolites (methionine sulfoxide, tryptophan, phenylalanine, alanine, and ornithine) and the increased concentration of 28 metabolites (21 phosphatidylcholines, 3 sphingomyelins, spermine, citrulline, acetylcarnitine, and hydroxybutyrylcarnitine) in hypothermia fatalities compared to controls. The signature shows similarities with already identified features in serum such as the altered concentrations of tryptophan, acylcarnitines, and unsaturated phosphatidylcholines, revealing a highly significant increased activity of methionine sulfoxide reductase, attested by a low methionine sulfoxide-to-methionine ratio. Our results show a preliminary metabolomics signature of hypothermia fatalities in the vitreous humor, highlighting an increased methionine sulfoxide reductase activity.