A circadian-like gene network programs the timing and dosage of heterochronic miRNA transcription during C. elegans development.

Development relies on the exquisite control of both the timing and the levels of gene expression to achieve robust developmental transitions. How cis- and trans-acting factors control both aspects simultaneously is unclear. We show that transcriptional pulses of the temporal patterning microRNA (miRNA) lin-4 are generated by two nuclear hormone receptors (NHRs) in C. elegans, NHR-85 and NHR-23, whose mammalian orthologs, Rev-Erb and ROR, function in the circadian clock. Although Rev-Erb and ROR antagonize each other to control once-daily transcription in mammals, NHR-85/NHR-23 heterodimers bind cooperatively to lin-4 regulatory elements to induce a single pulse of expression during each larval stage. Each pulse's timing, amplitude, and duration are dictated by the phased expression of these NHRs and the C. elegans Period ortholog, LIN-42, that binds to and represses NHR-85. Therefore, during nematode temporal patterning, an evolutionary rewiring of circadian clock components couples the timing of gene expression to the control of transcriptional dosage.

An Epigenetic Priming Mechanism Mediated by Nutrient Sensing Regulates Transcriptional Output during C. elegans Development.

Although precise tuning of gene expression levels is critical for most developmental pathways, the mechanisms by which the transcriptional output of dosage-sensitive molecules is established or modulated by the environment remain poorly understood. Here, we provide a mechanistic framework for how the conserved transcription factor BLMP-1/Blimp1 operates as a pioneer factor to decompact chromatin near its target loci during embryogenesis (hours prior to major transcriptional activation) and, by doing so, regulates both the duration and amplitude of subsequent target gene transcription during post-embryonic development. This priming mechanism is genetically separable from the mechanisms that establish the timing of transcriptional induction and functions to canalize aspects of cell-fate specification, animal size regulation, and molting. A key feature of the BLMP-1-dependent transcriptional priming mechanism is that chromatin decompaction is initially established during embryogenesis and maintained throughout larval development by nutrient sensing. This anticipatory mechanism integrates transcriptional output with environmental conditions and is essential for resuming normal temporal patterning after animals exit nutrient-mediated developmental arrests.

HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis.

Stochastic mechanisms diversify cell fate in organisms ranging from bacteria to humans [1-4]. In the anchor cell/ventral uterine precursor cell (AC/VU) fate decision during C. elegans gonadogenesis, two "α cells," each with equal potential to be an AC or a VU, interact via LIN-12/Notch and its ligand LAG-2/DSL [5, 6]. This LIN-12/Notch-mediated interaction engages feedback mechanisms that amplify a stochastic initial difference between the two α cells, ensuring that the cell with higher lin-12 activity becomes the VU while the other becomes the AC [7-9]. The initial difference between the α cells was originally envisaged as a random imbalance from "noise" in lin-12 expression/activity [6]. However, subsequent evidence that the relative birth order of the α cells biases their fates suggested other factors may be operating [7]. Here, we investigate the nature of the initial difference using high-throughput lineage analysis [10]; GFP-tagged endogenous LIN-12, LAG-2, and HLH-2, a conserved transcription factor that orchestrates AC/VU development [7, 11]; and tissue-specific hlh-2 null alleles. We identify two stochastic elements: relative birth order, which largely originates at the beginning of the somatic gonad lineage three generations earlier, and onset of HLH-2 expression, such that the α cell whose parent expressed HLH-2 first is biased toward the VU fate. We find that these elements are interrelated, because initiation of HLH-2 expression is linked to the birth of the parent cell. Finally, we provide a potential deterministic mechanism for the HLH-2 expression bias by showing that hlh-2 is required for LIN-12 expression in the α cells.

Glutamate spillover in C. elegans triggers repetitive behavior through presynaptic activation of MGL-2/mGluR5.

Glutamate is a major excitatory neurotransmitter, and impaired glutamate clearance following synaptic release promotes spillover, inducing extra-synaptic signaling. The effects of glutamate spillover on animal behavior and its neural correlates are poorly understood. We developed a glutamate spillover model in Caenorhabditis elegans by inactivating the conserved glial glutamate transporter GLT-1. GLT-1 loss drives aberrant repetitive locomotory reversal behavior through uncontrolled oscillatory release of glutamate onto AVA, a major interneuron governing reversals. Repetitive glutamate release and reversal behavior require the glutamate receptor MGL-2/mGluR5, expressed in RIM and other interneurons presynaptic to AVA. mgl-2 loss blocks oscillations and repetitive behavior; while RIM activation is sufficient to induce repetitive reversals in glt-1 mutants. Repetitive AVA firing and reversals require EGL-30/Gαq, an mGluR5 effector. Our studies reveal that cyclic autocrine presynaptic activation drives repetitive reversals following glutamate spillover. That mammalian GLT1 and mGluR5 are implicated in pathological motor repetition suggests a common mechanism controlling repetitive behaviors.

RAB-35 and ARF-6 GTPases Mediate Engulfment and Clearance Following Linker Cell-Type Death.

Clearance of dying cells is essential for development and homeostasis. Conserved genes mediate apoptotic cell removal, but whether these genes control non-apoptotic cell removal is a major open question. Linker cell-type death (LCD) is a prevalent non-apoptotic developmental cell death process with features conserved from C. elegans to vertebrates. Using microfluidics-based long-term in vivo imaging, we show that unlike apoptotic cells, the C. elegans linker cell, which dies by LCD, is competitively phagocytosed by two neighboring cells, resulting in cell splitting. Subsequent cell elimination does not require apoptotic engulfment genes. Rather, we find that RAB-35 GTPase is a key coordinator of competitive phagocytosis onset and cell degradation. RAB-35 binds CNT-1, an ARF-6 GTPase activating protein, and removes ARF-6, a degradation inhibitor, from phagosome membranes. This facilitates phosphatidylinositol-4,5-bisphosphate removal from phagosome membranes, promoting phagolysosome maturation. Our studies suggest that RAB-35 and ARF-6 drive a conserved program eliminating cells dying by LCD.

Post-mortem review of fentanyl-related overdose deaths among identified drug users in Southern Bavaria, Germany, 2005-2014.

BACKGROUND: Two decades ago, there were only single case reports on deaths in Europe following the consumption of illicitly manufactured fentanyl by problem drug users. Today, lethal fentanyl intoxication is now no longer a rarity. Since 2005, a rapid increase of lethal fentanyl-related intoxications in the drug scene has been observed at the Institute of Legal Medicine, Ludwig Maximilians University, Munich. We hypothesized that this rise is the result of the launch of fentanyl matrix patches in Germany in 2004, their broad acceptance, their diversion from the regulated supply chain, and incautious prescription by medical care providers. METHODS: Post-mortem toxicological reports were reviewed for lethal fentanyl-related intoxications between 2004 und 2014. Blood and tissue samples were tested by GC/MS or LC-MS/MS. The results of police investigations, autopsy reports, and the database of the Institute of Legal Medicine, LMU, were analysed to identify problem drug users and to detect the source of fentanyl as well as the routes of administration. RESULTS: Between 2005 and 2014, 242 overdose victims with post-mortem toxicological detection of fentanyl were found. In the majority of cases, fentanyl matrix patches were the source of fentanyl. CONCLUSION: The onset of fentanyl-related deaths coincided with the launch of transdermal fentanyl matrix patches in Germany in 2004. Several approaches, such as providing drug users with information on the possible risks of fentanyl consumption, education of medical caregivers, and also monitoring of the prescription of fentanyl patches, are required to reduce the number of fentanyl-related deaths in drug addicts.

Long-Term High-Resolution Imaging of Developing C. elegans Larvae with Microfluidics.

Long-term studies of Caenorhabditis elegans larval development traditionally require tedious manual observations because larvae must move to develop, and existing immobilization techniques either perturb development or are unsuited for young larvae. Here, we present a simple microfluidic device to simultaneously follow development of ten C. elegans larvae at high spatiotemporal resolution from hatching to adulthood (∼3 days). Animals grown in microchambers are periodically immobilized by compression to allow high-quality imaging of even weak fluorescence signals. Using the device, we obtain cell-cycle statistics for C. elegans vulval development, a paradigm for organogenesis. We combine Nomarski and multichannel fluorescence microscopy to study processes such as cell-fate specification, cell death, and transdifferentiation throughout post-embryonic development. Finally, we generate time-lapse movies of complex neural arborization through automated image registration. Our technique opens the door to quantitative analysis of time-dependent phenomena governing cellular behavior during C. elegans larval development.

Concentrations of cyanide in blood samples of corpses after smoke inhalation of varying origin.

Cyanide (CN) blood concentration is hardly considered during routine when evaluating smoke gas intoxications and fire victims, although some inflammable materials release a considerable amount of hydrogen cyanide. CN can be significant for the capacity to act and can in the end even be the cause of death. Systematic data concerning the influence of different fire conditions, especially those of various inflammable materials, on the CN-blood concentration of deceased persons do not exist. This study measured the CN level in 92 blood samples of corpses. All persons concerned were found dead in connection with fires and/or smoke gases. At the same time, the carboxyhemoglobin (COHb) level was determined, and the corpses were examined to detect pharmaceutical substances, alcohol and drugs. Furthermore, we analysed autopsy findings and the investigation files to determine the inflammable materials and other circumstances of the fires. Due to the inflammable materials, the highest concentration of CN in the victims was found after enclosed-space fires (n = 45) and after motor-vehicle fires (n = 8). The CN levels in these two groups (n = 53) were in 47 % of the cases toxic and in 13 % of the cases lethal. In victims of charcoal grills (n = 17) and exhaust gases (n = 6), no or only traces of CN were found. Only one case of the self-immolations (n = 12) displayed a toxic CN level. The results show that CN can have considerable significance when evaluating action ability and cause of death with enclosed-space fires and with motor-vehicle fires.

An autopsy approach to bolus deaths.

BACKGROUND: The term "bolus death" or "cafe coronary" refers to a misplaced larynx or laryngeal inlet by foreign bodies, in most cases by unchewed pieces of food such as meat, sausage, or fruit. The pathophysiologic mechanism of death is still debatable - sudden reflex cardiac arrests and asphyxial suffocations are implicated. In particular, children, alcoholics, and persons with brain-related damages belong to this risk group. A defective dentition is also associated with the risk of bolus deaths. AIM: The aim of the study was to supplement existing literature by evaluating cases of bolus deaths and determining the most likely mechanism of death in a large autopsy sample. MATERIAL: Among 31,647 autopsies, 59 cases of cafe coronary syndrome were identified and evaluated: 38 male adults and 21 female adults between the ages of 26 and 89 years. METHODS: A retrospective analysis of autopsies performed in the Institutes of Forensic Medicine in Rostock (1990-2014) and Munich (1990-1999) was conducted. Autopsy findings, circumstances of death, police investigations, preexisting conditions, dentition, and toxicological investigations were recorded. Then, based on the presence or absence of selected findings and surrounding circumstances, conclusions on the mechanism of death were drawn. RESULTS: In eighty-six percent (86%), boluses of meat and sausage or meat-like chunks were observed. Of the forty-nine victims, 22 (42%) had elevated blood alcohol levels (>0.5‰) with blood alcohol concentration (BAC) levels of >0.73‰ to <3.99‰. Of the nonalcoholized decedents, 63% had neurological disorders and 11% had preexisting cardiac conditions. CONCLUSIONS: Detecting potential bolus events upon autopsy and the resulting mechanism of nonnatural deaths are of great importance. Therefore, the medical history, resuscitation protocol, and detailed description of the scene of death are essential. Upon autopsy, the type and position as well as size and weight of the bolus and dental status of the victims should be documented in a standardized manner. Additional toxicological investigations are necessary.

Random Wiring, Ganglion Cell Mosaics, and the Functional Architecture of the Visual Cortex.

The architecture of iso-orientation domains in the primary visual cortex (V1) of placental carnivores and primates apparently follows species invariant quantitative laws. Dynamical optimization models assuming that neurons coordinate their stimulus preferences throughout cortical circuits linking millions of cells specifically predict these invariants. This might indicate that V1's intrinsic connectome and its functional architecture adhere to a single optimization principle with high precision and robustness. To validate this hypothesis, it is critical to closely examine the quantitative predictions of alternative candidate theories. Random feedforward wiring within the retino-cortical pathway represents a conceptually appealing alternative to dynamical circuit optimization because random dimension-expanding projections are believed to generically exhibit computationally favorable properties for stimulus representations. Here, we ask whether the quantitative invariants of V1 architecture can be explained as a generic emergent property of random wiring. We generalize and examine the stochastic wiring model proposed by Ringach and coworkers, in which iso-orientation domains in the visual cortex arise through random feedforward connections between semi-regular mosaics of retinal ganglion cells (RGCs) and visual cortical neurons. We derive closed-form expressions for cortical receptive fields and domain layouts predicted by the model for perfectly hexagonal RGC mosaics. Including spatial disorder in the RGC positions considerably changes the domain layout properties as a function of disorder parameters such as position scatter and its correlations across the retina. However, independent of parameter choice, we find that the model predictions substantially deviate from the layout laws of iso-orientation domains observed experimentally. Considering random wiring with the currently most realistic model of RGC mosaic layouts, a pairwise interacting point process, the predicted layouts remain distinct from experimental observations and resemble Gaussian random fields. We conclude that V1 layout invariants are specific quantitative signatures of visual cortical optimization, which cannot be explained by generic random feedforward-wiring models.

Can retinal ganglion cell dipoles seed iso-orientation domains in the visual cortex?

It has been argued that the emergence of roughly periodic orientation preference maps (OPMs) in the primary visual cortex (V1) of carnivores and primates can be explained by a so-called statistical connectivity model. This model assumes that input to V1 neurons is dominated by feed-forward projections originating from a small set of retinal ganglion cells (RGCs). The typical spacing between adjacent cortical orientation columns preferring the same orientation then arises via Moiré-Interference between hexagonal ON/OFF RGC mosaics. While this Moiré-Interference critically depends on long-range hexagonal order within the RGC mosaics, a recent statistical analysis of RGC receptive field positions found no evidence for such long-range positional order. Hexagonal order may be only one of several ways to obtain spatially repetitive OPMs in the statistical connectivity model. Here, we investigate a more general requirement on the spatial structure of RGC mosaics that can seed the emergence of spatially repetitive cortical OPMs, namely that angular correlations between so-called RGC dipoles exhibit a spatial structure similar to that of OPM autocorrelation functions. Both in cat beta cell mosaics as well as primate parasol receptive field mosaics we find that RGC dipole angles are spatially uncorrelated. To help assess the level of these correlations, we introduce a novel point process that generates mosaics with realistic nearest neighbor statistics and a tunable degree of spatial correlations of dipole angles. Using this process, we show that given the size of available data sets, the presence of even weak angular correlations in the data is very unlikely. We conclude that the layout of ON/OFF ganglion cell mosaics lacks the spatial structure necessary to seed iso-orientation domains in the primary visual cortex.

Deaths due to physical restraint.

BACKGROUND: Physical restraint is used primarily for patients at risk of falling, those with motor unrest and agitated behavior, and those who manifest an intention of doing harm to themselves or are at risk of suicide. The use of freedom-restraining measures (FRM), and, in particular, the use of physical restraints against the patient's will, can be a serious intrusion of basic human rights and, as such, an act of violence against the patient. The improper use of physical restraints can cause injuries of varying severity, which can sometimes be fatal. METHODS: We analyzed all cases of death under physical restraint that were recorded in the autopsy reports of the Institute of Forensic Medicine in Munich from 1997 to 2010. RESULTS: Among the 27 353 autopsies conducted over the period of the study, there were 26 cases of death while the individual was physically restrained. Three of these cases involved patients who died of natural causes while restrained, and one was a suicide. The remaining 22 deaths were caused solely by physical restraint; all of them occurred in patients under nursing care who were not continuously observed. The immediate cause of death was strangulation (11 cases), chest compression (8 cases), or dangling in the head-down position (3 cases). In 19 of these 22 patients, the restraints were incorrectly fastened, including two cases in which improvised non-standard restraints were used. One nursing-home patient died because of an abdominal restraint even though it had been correctly applied: She was mobile enough to slip through the restraint till it compressed her neck, and then unable to extricate herself from it, so that she died of strangulation. CONCLUSION: To prevent such deaths, we recommend from a forensic medical standpoint that all possible alternatives to FRM should be used instead. If direct-contact restraints are truly necessary, they must be applied as recommended and the restrained person must be closely observed.

Coverage, continuity, and visual cortical architecture.

BACKGROUND: The primary visual cortex of many mammals contains a continuous representation of visual space, with a roughly repetitive aperiodic map of orientation preferences superimposed. It was recently found that orientation preference maps (OPMs) obey statistical laws which are apparently invariant among species widely separated in eutherian evolution. Here, we examine whether one of the most prominent models for the optimization of cortical maps, the elastic net (EN) model, can reproduce this common design. The EN model generates representations which optimally trade of stimulus space coverage and map continuity. While this model has been used in numerous studies, no analytical results about the precise layout of the predicted OPMs have been obtained so far. RESULTS: We present a mathematical approach to analytically calculate the cortical representations predicted by the EN model for the joint mapping of stimulus position and orientation. We find that in all the previously studied regimes, predicted OPM layouts are perfectly periodic. An unbiased search through the EN parameter space identifies a novel regime of aperiodic OPMs with pinwheel densities lower than found in experiments. In an extreme limit, aperiodic OPMs quantitatively resembling experimental observations emerge. Stabilization of these layouts results from strong nonlocal interactions rather than from a coverage-continuity-compromise. CONCLUSIONS: Our results demonstrate that optimization models for stimulus representations dominated by nonlocal suppressive interactions are in principle capable of correctly predicting the common OPM design. They question that visual cortical feature representations can be explained by a coverage-continuity-compromise.

Reorganization of columnar architecture in the growing visual cortex.

Many cortical areas increase in size considerably during postnatal development, progressively displacing neuronal cell bodies from each other. At present, little is known about how cortical growth affects the development of neuronal circuits. Here, in acute and chronic experiments, we study the layout of ocular dominance (OD) columns in cat primary visual cortex during a period of substantial postnatal growth. We find that despite a considerable size increase of primary visual cortext, the spacing between columns is largely preserved. In contrast, their spatial arrangement changes systematically over this period. Whereas in young animals columns are more band-like, layouts become more isotropic in mature animals. We propose a novel mechanism of growth-induced reorganization that is based on the "zigzag instability," a dynamical instability observed in several inanimate pattern forming systems. We argue that this mechanism is inherent to a wide class of models for the activity-dependent formation of OD columns. Analyzing one representative of this class, the Elastic Network model, we show that this mechanism can account for the preservation of column spacing and the specific mode of reorganization of OD columns that we observe. We conclude that column width is preserved by systematic reorganization of neuronal selectivities during cortical expansion and that this reorganization is well described by the zigzag instability. Our work suggests that cortical circuits may remain plastic for an extended period in development to facilitate the modification of neuronal circuits to adjust for cortical growth.

Deaths following methotrexate overdoses by medical staff.

Methotrexate (MTX) is an effective disease modifying antirheumatic drug (DMARD) with a relatively safe profile, and it is widely used to treat neoplastic diseases and dermatologic and rheumatologic disorders. As indications for use of MTX increase, more accidental overdoses are noted to occur. Typical problems include deficiencies in labeling, instructions, or packaging, as well as erroneous use. We describe 5 fatal cases of repeated oral overdose of MTX prescribed by physicians in the treatment of rheumatoid arthritis to focus attention on the design of the underlying system and the organizational practices as sources of problems.

Do centenarians die healthy? An autopsy study.

BACKGROUND: Our goal was to assess the prevalence of common causes of death and the demographic variables in a selected population of centenarians. METHODS: The autopsy reports and medical histories of all individuals > or =100 years, dying unexpectedly out of hospital, were gathered from 42,398 consecutive autopsies, performed over a period of 18 years at the Institute of Forensic Medicine, Vienna. These records were evaluated with regard to age and sex, circumstances of death, season, time and the cause of death, as well as the presence of any other comorbidity. RESULTS: Forty centenarians (11 men, 29 women) were identified with a median age of 102 +/- 2.0 (range: 100-108) years. Sixty percent were described as having been healthy before death. However, an acute organic failure causing death was found in 100%, including cardiovascular diseases in 68%, respiratory illnesses in 25%, gastrointestinal disorders in 5%, and cerebrovascular disease in 2%. Additionally, centenarians suffered from several comorbidities (cardiac antecedents, neurologic disorders, liver diseases, cholecystolithiasis), which were not judged to be the cause of death. CONCLUSIONS: Centenarians, though perceived to have been healthy just prior to death, succumbed to diseases in 100% of the cases examined. They did not die merely "of old age." The 100% post mortem diagnosis of death as a result of acute organic failure justifies autopsy as a legal requirement for this clinically difficult age group.

Application of the BioRobot EZ1 in a forensic laboratory.

In a forensic laboratory, the routine application of an automated DNA extraction and purification robot has to fulfil several conditions, like producing reproducible DNA's of sufficient quantity and quality from all the different forensic biological stains relevant to various carrier materials. In this study, the suitability of the BioRobot EZ1 system from QIAGEN (Hilden, Germany), which offers fully automated extraction and purification of nucleic acids using magnetic bead technology, was tested. In summary, the DNA's obtained from the BioRobot EZ1 for different forensic relevant biological materials showed a quantity and quality comparable to those of the forensic standard protocols normally used in our laboratory. The system saves time, because there is no need of any further purification or concentration step after the automated DNA extraction. It can also be used as a replacement for time consuming organic extractions. A disadvantage of the system was the unsteady quality of the chemical regencies used by the robot. Nineteen different lots were tested with a self designed test system.

Foreign body asphyxia: a preventable cause of death in the elderly.

BACKGROUND: To assess the prevalence of food/foreign body asphyxia in the elderly Viennese population in order to reduce the incidence of these fatal events. METHODS: This is an autopsy-based, retrospective study in Vienna, Austria. Participants included all nonhospitalized (n =200) cases of choking in 1984 to 2001, from a total 42,745 consecutive autopsies performed at the Institute of Forensic Medicine. In addition, data from hospitalized adult cases of fatal choking (n =73) in 1984 to 2001, from the mortality registrar of Vienna, were included. RESULTS: The nonhospitalized choking victims were analyzed according to age (18 to 64 vs >/=65 years), sex, circumstances of death, and predisposing factors. Hospitalized cases were analyzed according to age, sex, and whether an autopsy was already performed by pathologists at the institution where they died. In the study period, 273 adults died of food/foreign body asphyxia, 73% of them out of the hospital and 27% in hospitals. Food/foreign body asphyxia in the elderly was characterized by a significantly higher asphyxiation of soft/slick foods (p <0.007) with agomphiasis (p <0.002), occurring most frequently during lunch (49%), and in 2.5% during feeding of neurologically impaired. In contrast, younger individuals choked significantly more often on large pieces of foreign material (p <0.002) and showed a significantly higher rate of blood alcohol concentration (p <0.001). CONCLUSIONS: This study demonstrates that semisolid foods are the cause of a large number of asphyxiations, especially among the elderly. Knowledge of the fact that semisolid foods are a high-risk factor in elderly individuals should be distributed in public and private healthcare systems, and awareness could be a first step in reducing the incidence of food/foreign body asphyxia.

Investigation of oral swabs taken from newborn infants after breastfeeding.

The aim of this study was to investigate the influence of breastfeeding on the DNA-profiles from oral swabs of newborn babies. Five mother and child pairs were asked to provide orals swabs from the mother and from the child before and immediately after nursing. Investigation of the samples revealed no maternal alleles in the saliva samples of the child taken directly after breast feeding. Therefore, we conclude that nursing does not influence the STR-typing of oral swabs taken form newborn infants. A possible explanation could be that the DNA content of mother's milk is significantly lower than the DNA content of saliva.