Lateral septum adenosine A2A receptors control stress-induced depressive-like behaviors via signaling to the hypothalamus and habenula.

Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.

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.

Lateral septum adenosine A2A receptors control stress-induced depressive-like behaviors via signal-ing to hypothalamus and habenula / 中国药理学与毒理学杂志

Depressive disorder ranks as a major bur-den of disease worldwide,yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects.The lateral septum(LS)is thought to control of depression,however,the cellular and circuit substrates are largely unknown.Here,we identified a subpopulation of LS GABAergic adenosine A2A receptors(A2AR)-positive neurons mediating depres-sive symptoms via direct projects to the lateral habenula(LHb)and the dorsomedial hypothalamus(DMH).Activa-tion of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipula-tion of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive pheno-types.Thus,the optogenetic modulation(stimulation or inhibition)of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH,phenocopied depressive behaviors.Moreover,A2AR are upregulated in the LS in two male mouse mod-els of repeated stress-induced depression.This identifica-tion that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant poten-tial of A2AR antagonists,prompting their clinical transla-tion.

Survey on postmortem screening and management of COVID-19 related deaths.

The COVID-19 pandemic is associated with a high case fatality rate in some countries even thought the majority of cases are asymptomatic. Scientific studies on this novel virus is limited and there is uncertainty regarding the best practices for death investigations both in terms of detection of the disease as well as autopsy safety. An online survey was conducted to identify how different institutions responded to the screening and management of dead bodies during the early phase of the pandemic from January to May. A questionnaire was developed using Google Forms and data was collected from 14 different forensic and pathological institutions in 9 countries. None of the institutions had performed any screening prior to March. Four institutions stated that screening was done routinely. In total, 322 cases had been screened using RT-PCR, out of which 40 positive cases were detected among four institutions. The commonest types of samples obtained were nasopharyngeal and oropharyngeal swabs which also had the highest rates of positivity followed by tracheal swab. Blood, swabs from cut surfaces of lung and lung tissue also gave positive results in some cases. Majority of the positive cases were > 65 years with a history suggestive of respiratory infection and were clinically suspected to have COVID-19 before death. Except for one institution which performed limited dissections, standard autopsies were conducted on all positive cases. Disposal of bodies involved the use of sealed body bags and labelling as COVID positive. Funeral rites were restricted and none of the institutions advocated cremation. There were no reports of disease transmission to those who handled COVID positive bodies.

DNA methylation age estimation in blood samples of living and deceased individuals using a multiplex SNaPshot assay.

Many studies in the forensic field have reported that analysis of DNA methylation is the most reliable method of predicting age. In a previous study, 5 CpG sites located in ELOVL2, FHL2, KLF14, C1orf132 and TRIM59 genes were tested for age prediction purposes in blood, saliva and buccal swab samples from Korean individuals using a multiplex methylation SNaPshot assay. The main goals of the present study were i) to replicate the same multiplex SNaPshot assay in blood samples from Portuguese individuals, ii) to compare DNA methylation status between two different populations and iii) to address putative differences in the methylation status between blood from living and deceased individuals. Blood samples from 59 living individuals (37 females, 22 males; aged 1-94 years-old) and from 62 deceased individuals (13 females, 49 males; aged 28-86 years-old) were evaluated. The specific primers were those previously described. Linear regression models were used to analyse relationships between methylation levels and chronological age using IBM SPSS software v.24. Our results allowed to build a final age prediction model (APM) for blood samples of living individuals with 3 CpG sites, at ELOVL2, FHL2 and C1orf132 genes, explaining 96.3% of age variation, with a mean absolute deviation (MAD) from chronological age of 4.25 years. Some differences were found in the extent of the age association in the targeted loci comparing Portuguese with Korean individuals. The final APM built for deceased individuals included 4 CpG sites, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explaining 79.3% of age variation, with a MAD of 5.36 years. Combining both sets of samples from living and deceased individuals, the most accurate APM with 4 CpGs, at ELOVL2, FHL2, C1orf132 and TRIM59 genes, explained 92.5% of variation in age, with a MAD of 4.97 years. In conclusion, our study replicated in blood samples of Portuguese living individuals a previous SNaPshot assay for age estimation. The possibility that age markers might be population specific and that postmortem changes can alter the methylation status among specific loci was suggested by our data. Our study showed the usefulness of the multiplex methylation SNaPshot assay for forensic analysis in blood samples of living and deceased individuals.

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals.

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.

A reliable method for estimating the postmortem interval from the biochemistry of the vitreous humor, temperature and body weight.

The estimation of the time elapsed since death is of paramount importance in the field of forensic sciences and criminal investigation, owing, among other factors, to the possible legal repercussions. Over the past few years various formulae have been developed to calculate this interval using a combination of different statistical methods and the concentrations of substances found in the vitreous humor. Corrective factors, such as ambient temperature, cause of death or age, which can modify the concentration of these substances and therefore the estimation of the postmortem interval, have been incorporated into models. In this paper five simple and reliable models to estimate PMI based the on the analysis of potassium, hypoxanthine and urea in the vitreous humor are presented. Corrective factors, such as body weight, rectal temperature and ambient temperature, which can influence the estimation of this interval have been incorporated into the formulae. Finally, the R2 and the mean squared error have been calculated for each model in order to select the best of the five. A free software program which calculates the PMI from the model and parameters used is available from the authors. It provides quick and reliable results as well as the error committed and R2 for each case.

Analysis of fatal accidents with tractors in the Centre of Portugal: Ten years analysis.

Tractors have been described as one of the deadliest farming implements concerning agricultural activity. In Portugal, the scientific investigations about this problem are practically non-existent, with only statistical studies performed by entities related to road traffic safety, not in accordance to the study now performed, pinpointing the possibility of an underreporting of these accidents. This work aims to characterize the fatal tractor accidents in Portugal, autopsied at the Forensic Pathology Department of the Centre Branch of the National Institute of Legal Medicine and Forensic Sciences of Portugal, analysing several variables: gender, age, occupation, survival time, the victim position in the tractor, cause of death, toxicological and histological exams, year/month/day of the week, type of agricultural machine, existence of rollover protective structures (ROPS), type of accident, ground conditions, circumstantial information and geographic distribution of the accidents. All the autopsies between 2005 and 2014 were analysed. The victim profile corresponded to a man (89.5%), between 61 and 70 years old (33.3%), retired (43.9%), being the tractor driver (45.6%). In most of the cases, death occurred in less than 24h after the accident. These fatalities arose mainly in May and October. Rollover in sloping land was the most common type of accident, and cranioencephalic, thoracic and abdominal traumatic injuries were the main cause of death. In 16.2% of the cases, blood alcohol concentration was above the lower limit established in our road traffic law (<0.5g/L). There was lack information about the use of ROPS (95.9%), and even when existent, the protections were not used or were incorrectly used. This is the first national study involving the description of the forensic achievements in each autopsy related to tractor accidents, and the corresponding circumstances that contributed to the death. Many barriers remain about this matter, but the Portuguese government begin to design several laws to prevent this type of accidents. Future investigations should be performed to understand the real dimension of tractor accidents in Portugal, and to comprehend the efficiency of laws implemented by the government.

Uranium accumulation in aquatic macrophytes in an uraniferous region: Relevance to natural attenuation.

Phytoremediation potential of uranium (U) was investigated by submerged, free-floating and rooted emergent native aquatic macrophytes inhabiting along the streams of Horta da Vilariça, a uraniferous geochemical region of NE Portugal. The work has been undertaken with the following objectives: (i) to relate the U concentrations in water-sediment-plant system; and (ii) to identify the potentialities of aquatic plants to remediate U-contaminated waters based on accumulation pattern. A total of 25 plant species culminating 233 samples was collected from 15 study points along with surface water and contiguous sediments. Concentrations of U showed wide range of variations both in waters (0.61-5.56 µg L(-1), mean value 1.98 µg L(-1)) and sediments (124-23,910 µg kg(-1), mean value 3929 µg kg(-1)) and this is also reflected in plant species examined. The plant species exhibited the ability to accumulate U several orders of magnitude higher than the surrounding water. Maximum U concentrations was recorded in the bryophyte Scorpiurium deflexifolium (49,639 µg kg(-1)) followed by Fontinalis antipyretica (35,771 µg kg(-1)), shoots of Rorippa sylvestris (33,837 µg kg(-1)), roots of Oenanthe crocata (17,807 µg kg(-1)) as well as in Nasturtium officinale (10,995 µg kg(-1)). Scorpiurium deflexifolium displayed a high bioconcentration factor (BF) of ∼2.5 × 10(4) (mean value). The species Fontinalis antipyretica, Nasturtium officinale (roots) and Rorippa sylvestris (shoots) exhibited the mean BFs of 1.7 × 10(4), 5 × 10(3) and 4.8 × 10(3) respectively. Maximum translocation factor (TF) was very much pronounced in the rooted perennial herb Rorippa sylvestris showing extreme ability to transport U for the shoots and seems to be promising candidate to be used as bioindicator species.

The Application of Flow Cytometry as a Rapid and Sensitive Screening Method to Detect Contamination of Vitreous Humor Samples and Avoid Miscalculation of the Postmortem Interval.

Research into maximizing the speed, precision, and reliability of estimating the postmortem interval (PMI) has been a recurring object of investigation and methodologies based on the vitreous humor (VH) have provided good results. However, contamination from causes not readily apparent, such as blood, can occur, and thus lead not only to an erroneous estimation of PMI, but also interfere with the correct identification of other substances in the VH. We have developed a flow cytometry method which quantifies blood contamination and is able to detect erythrocytes in 1:750,000 dilution of contaminated VH which affects the results of hypoxanthine. It is an improvement on the previous more complex mass spectrometry method, being faster, more sensitive, and readily available. As such, it could be proposed for the rapid screening of appropriate samples by detecting and eliminating blood contaminated samples from PMI estimation.

High variation in hypoxanthine determination after analytical treatment of vitreous humor samples.

In the field of legal medicine the correct determination of the time of death is of great importance because an error in calculating the post-mortem interval (PMI) could be crucial in a criminal investigation. The quantification of hypoxanthine (Hx) concentration in the vitreous humor (VH) as a means to estimate PMI is useful when dealing with a recent time interval of death and has several advantages over other biological matrices. However, due to its viscous nature, VH has to be liquefied prior to its analysis, and the different procedures to liquefy it may cause alterations in Hx concentration, and hence in estimation of PMI. The aim of this study was to determine differences in Hx concentration after applying several pre-analytical treatments to different aliquots of the same VH sample. Enzymatic digestion, sonication, centrifugation and heat were the liquefying methods applied to reduce the viscosity of 105 VH samples. Analysis was performed using solid phase extraction and liquid chromatography in tandem with mass spectrometry. Statistically significant differences in the measured Hx concentrations were found between enzymatic digestion and the other three methods analyzed (sonication, centrifugation, and heat). Additionally, high data dispersion was found under heat treatment but was not observed for centrifugation or sonication, where similar and more moderate results were found. We conclude that high variations in Hx determination are observed when enzymatic digestion or heat treatment is used and we therefore recommend sonication or centrifugation to quantify Hx concentration in the VH.

The use of computerized tomography in determining stature and sex from metatarsal bones.

This study evaluates the efficacy of a radiological method to estimate stature from measurements of the first and second metatarsal taken from a collection of metatarsals of a Portuguese Caucasian population in which the measurements were made directly on the bone. The highest coefficient of determination and the lowest standard error were obtained with the physiological length of the second metatarsal (F2), using the equation S=895.4803+10.7848F2. The linear regressions obtained show significant differences between the estimated heights from M1. In addition, we offer a simple method for sex determination based on the maximum length (M1) and width (W1) of the first metatarsal, where W1=x1; (M1/W1)=x2; ß0=55.4767; ß1=-2.5796 and ß2=-4.6898. Here we present a method of measurement using computerized tomography that enables population studies using live volunteers without incurring the difficulties of on the bone measurement.

Unusual Forms of Adrenal and Extra-Adrenal Myelolipomas.

Myelolipomas are rare benign tumors of poorly understood tumorigenesis composed of mature hematopoietic tissue and fat. They mostly occur in the adrenal glands, but extra-adrenal myelolipomas have been reported in other locations such as the presacral region or retroperitoneum. It is not unusual that they are incidental findings revealed in the study of different diseases. We report 3 unusual examples of myelolipomas. The first is a multiple, unusually large, extra-adrenal myelolipoma, presented as an autopsy finding in an individual who had died suddenly from a central nervous system hemorrhage. The remaining 2 were incidental findings in patients studied for different reasons. Both were located within another neoplasm, namely an adrenal adenoma and a liver focal nodular hyperplasia. Moreover, the first showed infiltration by a non-Hodgkin lymphoma.

Cardiac inhibitory reflex as a cause/mechanism of death.

The role of cardiac inhibitory reflex as a potential cause of death is still a matter of debate. This study reports two cases of death under unusual circumstances. Case 1 corresponds to a man found hanging where the role of ligature compression of the carotid sinus became relevant as a possible explanation of death. In Case 2, the participation of a vasovagal syncope was clearly triggered by the laryngoscopic procedure. It is proposed that cardiac inhibitory reflex should be taken into account in those cases of unexpected death, which fulfills the following three criteria: (i) The investigation of the circumstances of the death is consistent with a hypothesis of cardiac arrest. (ii) A typical triggering peripheral stimulus is present. (iii) The performance of a complete autopsy cannot rule out the participation of a cardiac inhibitory reflex in the cause of death.

Applications of Tandem Mass Spectrometry (LC-MSMS) in estimating the post-mortem interval using the biochemistry of the vitreous humour.

It is widely accepted that the quantification of potassium concentrations ([K+]) and hypoxanthine ([Hx]) in the vitreous humour is useful in estimating the time of death within a recent time interval. Despite all the advances made in this area, it is well recognized that difficulties in calibration, validation and the use of different methodologies and instrumentation from different laboratories may lead to obtaining different concentrations from the same sample. The extraction of the vitreous humour itself should also be carried out with a precise technique, taking care during the procedure to avoid causing any vascular injury that might lead to haematic contamination. Any fluid that is not clear could lead to erroneous results and should be discarded. We present a new LC-MSMS method developed for quantitative and qualitative analysis of [Hx] (valid also for guanine and xanthine), and qualitative determination of uric acid in vitreous humour. We also introduce a methodology to assess haematic contamination in order to improve the estimation of time since death. The method was fully validated in terms of linearity, sensitivity, imprecision, analytical recovery, extraction and process efficiency and matrix effect.

Predicting adult stature from metatarsal length in a Portuguese population.

Stature can be considered one of the "big four" parameters to be ascertained within the biological profile in cases of forensic anthropology. However, the most reliable available methods for stature estimation require the preservation of the long bones, but since this is very often not the case, the development of alternative methods, based on distinct bones, is mandatory. Therefore, in the present work the reliability of the first two metatarsal bones in reconstructing stature is tested. The data consist of length measurements taken from the first two metatarsals removed from documented cadavers of known stature. The sample for this study consists of 220 metatarsals, namely 110 first metatarsals and 110 second metatarsals collected during the autopsies carried out in the National Institute of Legal Medicine in Portugal. The aim was to propose regression equations for the Portuguese population and test the formulae proposed by other authors to determine adult stature using metatarsal bones. We found that when estimating stature from measurement of the metatarsals, the best correlation was that obtained from the relationship with the maximum length of the 2nd metatarsal. The corresponding regression equation is as follows: S=790.041+11.689M2.

Male specific association between the 5-HTR6 gene 267C/T SNP and suicide in the Portuguese population.

Serotonergic system dysfunction has been implicated in the etiology of suicide. A large number of genetic studies have focused on the potential involvement of genes coding for components of serotonergic system in suicidal behavior. However, other genes belonging to this system remain to be investigated or have been poorly studied, as is the case of the 5-HT6 receptor (5-HTR6) gene. In this study, we investigated the potential association between the 5-HTR6 gene 267C/T SNP and suicide in a Portuguese population. Blood samples were collected from 179 suicide victims and 189 controls. Genotypes for the 5-HTR6 gene 267C/T SNP were obtained with the restriction enzyme Rsa I. A tendency was found for genotype association between this polymorphism and suicide, but the differences were not statistically significant (chi(2)=5.374, df=2, p=0.068). However, a gender-specific association was detected when comparing the genotype distribution between male suicide victims and male controls (chi(2)=6.988, df=2, p=0.030), suggesting that this SNP might have a role in the etiology of suicide in male subjects in the Portuguese population.

Characterization of lesions in hanging deaths.

Hanging is a common method of committing suicide and a routine task in medico-legal autopsies. The hanging mark is the most relevant external sign and its characteristics are well known, but, for unknown reasons, there are major differences in reports on internal findings. We retrospectively studied 228 consecutive cases of hanging deaths. A complete standard autopsy was performed for every case. We investigated the association between the characteristics of the hanging mark and the frequency of bone, cartilage, soft tissue, and vascular injuries with the mode of suspension. Most cases (75.3%) presented some kind of bone or cartilage fracture, but these were unrelated to any of the variables studied. Vascular lesions are clearly more infrequent: intimal injuries were found in the carotid artery (9.1%), the jugular vein (2.2%), and ruptures of the carotid adventitial layer (21.7%). These could be partially associated with the use of a hard fixed noose and body weight.