Philip Nicholls

Bone taphonomy and diagenesis contribute to anthropological analysis in forensic investigations by attempting to reconstruct the relationship between human cadaveric remains and their postmortem depositional environment. The rare aquatic taphonomic experiments have been delivering conflicting results on the influence of time and the environment on the decay of bone and teeth, especially considering that the main diagenetic processes can lead to fragmentation, progressive dissolution or fossilization. The aim of this experimental, quantitative, randomized and controlled 2-year study was to analyse the taphonomy and diagenesis of submerged terrestrial mammalian bones to achieve a more accurate estimation of both the post-mortem interval (PMI) and the post-mortem submersion interval (PMSI) in the short term. Three parameters of bone diagenesis, the Oxford Histological Index (OHI), the total porosity and the collagen content of cortical bone were analysed by MicroCT Scan, bright-field Light Microscopy (Picrosirius Red stain), Scanning Electron Microscopy (SEM) and Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) on 75 sheep femurs and tibias placed in four distinct types of environment (natural saltwater, natural freshwater, an artificial seawater solution and exposed to the air) vs. non-exposed controls. LA-ICP-MS was soon discontinued because no measurable changes of the elemental profiles could be detected. Multivariate statistical analysis was applied to the collected data. The macroscopical preservation was consistently excellent (OHI=5). The total porosity and the degradation of collagen were greater underwater than in subaerial exposure, whereas demineralization zones and bioerosion tunnelling appeared after 12 months in the air-exposed samples only. Underwater, the continuous movement, the correlated abrasion by sand and sediment and the constant alkaline pH (≥ 8) can explain the progressive removal of the mineral component and the subsequent exposure of collagen to bioeroders and chemical hydrolysis. On land, the same process occurs at a slower rate on account of the seasonality of the water flow, however, the action of the more abundant and diversified species of bioeroding microorganisms appears more efficient. Despite some limitations, this study indicates that three parameters of bone diagenesis can predict the depositional environment of terrestrial mammalian bone characterized by a PMI and/or PMSI of at least 12 months.

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (−) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.

Bone and teeth, specialised bio-mineralized connective tissues, are left after the typical decomposition process of any vertebrate organism. Their analysis can reveal insights into an organism's life and retrace the history of the remains after death (also known as taphonomy), which ultimately evolves to destruction or fossilization. Studies on the taphonomy of terrestrial mammalian bio-mineralized tissues have mostly focussed on terrestrial depositional environments. Here, samples submerged in the marine environment are investigated.

Five archaeological bones of terrestrial mammalian species (pig and oxen) with historically known post-mortem submersion interval (PMSI) (69–316 years) and recovery sites, were analysed macroscopically, microscopically and by microCT. The aim was to characterize for the first time the alterations produced by marine bioeroding sponges, and to discuss their potential interdisciplinary application, with special focus on forensic investigations.

The pig samples showed microanatomical preservation (Oxford Histological Index = 3–5), increased total porosity, the presence of old tissue flakes with sponge spicules and traces of bioerosion, such as papillary holes, canals and chambers with microsculptured walls. The presence of such tissue flakes suggested that, at the time of recovery, they may have been free of sediment and inhabited by live sponges. The shape of one internal chamber was identified as the ichnospecies Entobia convoluta as typically produced by shallow, warm-water Cliothosa spp. Surface analyses for further biological evidence remained inconclusive.

The taphonomy of skeletal remains has always been relevant in anthropological, natural and forensic studies. In forensics, the role of taphonomy is to contribute to personal identification, cause of death and post-mortem interval (PMI). This study detected the past colonization of terrestrial mammalian bone by marine bioeroding sponges, and aimed to link the taphonomic findings to natural processes and environments. Bioeroding sponges are for the first time confirmed to colonize terrestrial mammalian bone submerged in marine environments, and to promote diagenesis through bioerosion.

The central nervous system (CNS) influences the immune system generally by regulating the systemic concentration of humoral substances (e.g., cortisol and epinephrine), whereas the peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions/connections. An imbalance between the components of the PNS might contribute to pathogenesis and the further development of certain diseases. In this review, we have explored the “thread” (hardwiring) of the connections between the immune system (e.g., primary/secondary/tertiary lymphoid tissues/organs) and PNS (e.g., sensory, sympathetic, parasympathetic, and enteric nervous systems (ENS)) in health and disease in vitro and in vivo . Neuroimmune cell units provide an anatomical and physiological basis for bidirectional crosstalk between the PNS and the immune system in peripheral tissues, including lymphoid tissues and organs. These neuroimmune interactions/modulation studies might greatly contribute to a better understanding of the mechanisms through which the PNS possibly affects cellular and humoral-mediated immune responses or vice versa in health and diseases. Physical, chemical, pharmacological, and other manipulations of these neuroimmune interactions should bring about the development of practical therapeutic applications for certain neurological, neuroimmunological, infectious, inflammatory, and immunological disorders/diseases.

Unlike the chemical composition and diagenetic modification of buried bones, subaqueous archaeological bone diagenesis has not been studied in detail. This observational work presents a macroscopic and microscopic characterization of 11 variably preserved archaeological terrestrial mammalian bones submerged in seawater and/or surrounded by marine sediment for 169–347 years. In situ trace element analysis was undertaken to identify geochemical fingerprints of diagenesis. The analyzed bones belong to a collection of underwater archaeological faunal materials excavated from four shipwreck sites. With one exception, all archaeological bones were fragmented, some were also heavily stained, and in two samples, the damage to the cortical layer was extensive. Bioerosion was assessed by scanning electron microscopy (SEM), and bone trace element chemistry (by laser ablation inductively coupled mass spectrometry—LA-ICP-MS) was compared with that of an unsubmerged modern sheep bone control. In the control, several trace elements were low in concentration (weighted mean concentration <1 ppm; Cr, Co, Ni, Cu, Y, rare earth elements, Th, U). In the submerged archaeological bones, the weighted mean concentration of Li, Cr, Cu, and U was enriched relative to the modern sheep bone, whereas Rb and Ba were depleted. The best-preserved bone, recovered from Batavia, showed less variation in trace element patterns compared with the more poorly preserved bones. The only archaeological bone with preserved macroscopic structure and cortex showed a gradual decrease in trace element concentration from the outer surface towards the medullary cavity, whereas in samples where more cortical damage was noted, the distribution of these elements is more irregular. With the exception of Cu and Cr, the elements focused on in this work (Li, U, Rb, and Ba) are nonessential to life, supported by their low concentration in the modern sheep bone (with the exception of Ba). The results suggest that early macroscopic and microscopic diagenetic alteration influences the concentration and distribution of chemical elements in submerged bones and that in situ trace element analysis provides clues for the reconstruction of taphonomic trajectories.

Foraminifera are marine single-celled organisms, ubiquitous in marine environments, present in brackish waters and absent in terrestrial locations. Their presence has been associated with archaeological and forensic studies only rarely, and just once and superficially with bones of terrestrial mammals. In this study, a new association is presented between foraminifera enclosed in the dissolving trabecular spaces of terrestrial mammalian bones, recovered in underwater archaeological excavations between 1968 and 1980. Research on the new association aims to detail the micro-characterization of bone in underwater environments, leading to a better understanding of bone taphonomic trajectories, the chronological sequences of changes occurring between death and the incorporation of the remains of an organism within the depositional environment. The analysis of taphonomic trajectories is known to hold relevance in distinct disciplines, such as archaeology, palaeontology and forensic sciences. Different foraminiferal taxa are linked to different marine environments, characterized by specific ranges of water depth, amount of light and oxygen, temperature and composition of sediment. The association between foraminifera and terrestrial mammalian bones indicates deposition in a marine or brackish environment, thus the analysis of the specific ecology of the identified foraminiferal taxa can point to a specific environment, adding information to paleontological, archaeological or forensics casework.

Learning Overview: The goal of this presentation is to describe the principles of bone diagenesis and their potential application to forensic science, the distinction between biogenic and diagenetic chemical signals in bones, the correlation between macroscopic and chemical characteristics of bone diagenesis, and an example of trace element analysis in a set of archaeological bones submerged for a known length of time in a marine environment. Impact on the Forensic Science Community: This presentation will impact the forensic science community by describing how the establishment of a correlation between macroscopic alterations and trace element concentrations and distributions in bones recovered from a marine environment will prove essential for members of the forensic science community involved in taphonomic assessments. Bone diagenesis is the global effect of the physical, biological, and chemical transformations that bones undergo between death and discovery in the archaeological or geological record. Diagenetic transformations, macroscopic and microscopic, are influenced by the physics, chemistry, and biology of the depositional environment. In living organisms, chemical processes are affected by diet, mobility, and pathologies. While studies of the diagenetic modifications and chemical composition of buried bones are extensively featured in the scientific literature, geochemical signatures characteristic of underwater bone diagenesis have not been studied in detail. This study investigated whether a geochemical fingerprint of the interaction between 11 archaeological mammalian bones and seawater and/or marine sediment could be discerned. The analyzed mammalian bones belong to a museum collection of underwater archaeological materials excavated from four submerged shipwreck sites off the Western Australia coast: Batavia (1629), Vergulde Draeck (1656), Zeewijk (1727) and Rapid (1811). The underwater excavations were conducted between 1968 and 1980, and bones from the four wrecks had been submerged in seawater and/or sediment for 347, 316, 241, and 169 years, respectively. With one exception, all of archaeological bones were fragmented, some were also heavily stained, and in two samples, the damage to the protective cortical layer was particularly extensive. Bone trace element chemistry was compared to that of a modern sheep bone (Ovis aries). Laser ablation-inductively coupled plasma/mass spectrometry was undertaken across bones mounted in epoxy rounds. Cross-sectional spot transverses followed a path from the cortical layer (exterior) through the trabecular bone in the interior. In the modern sheep bone, several trace elements showed bulk concentrations close to, or at, the limit of detection (Chromium [Cr], Cobalt [Co], Nickel [Ni], Copper [Cu], Yttrium[Y], Rare-Earth Element [REE], Thorium [Th], and Uranium [U]). In contrast, in the submerged bones, Lithium (Li), Cr, Cu, and U were elevated relative to the modern sheep bone, whereas Rubidium (Rb) and Barium (Ba) were depleted. Normalized trace element patterns in modern bone were flat, whereas in the archaeological samples, the normalized trace element pattern in the only whole sample (from Batavia) was different from that of the damaged bones from the other wrecks. Most elements with altered bulk concentrations in the archaeological bones are non-essential to biological life (Cu being the exception), supported by their low concentration in the modern sheep bone. However, Ba is usually enriched in bone by reason of known para-physiological metabolic processes. Since Li, Cr, Cu, U, Rb, and Ba are present in seawater in very low concentrations (<1ppm), it is reasonable to assume that in the archaeological bones, the relevant increase in bulk concentrations of Li, Cr, Cu, and U is entirely diagenetic in origin, perhaps due to protracted chemical exchange with sediment. The depletion of bulk concentrations of Rb and Ba is also diagenetic in origin and can be explained by protracted exposure to seawater and sediment. Furthermore, since the structure of cortical bone is denser than that of trabecular bone, cortical bone is less susceptible to alteration. This is reflected in the flat normalized element distribution profiles in bones where the cortical layer is missing or heavily damaged. As a consequence, the bulk chemical composition resulting from diagenetic chemical exchange in bone appears to be more uniformly distributed if the cortical layer is heavily damaged or missing, as reflected by the flat normalized elemental distribution profiles. In the only undamaged sample, the profiles of Li, Ba, Magnesium (Mg), Strontium (Sr), and Rb showed a gradual decrease in concentration from the outer surface toward the interior of the cortical bone. The overall conclusion is that macroscopic diagenetic alterations influence elemental concentrations and patterns of elemental distribution in bones, and their analysis allows the reconstruction of different taphonomic pathways.

Diagenesis is the collective word for the physical, biological, and chemical processes that bones undergo in the post-mortem period, until their physical destruction or fossilization. In forensic anthropology, the analysis of macroscopic and microscopic bone alterations, alongside the taphonomy of the soft tissues of a body, has proven valuable for the estimation of the time-of-death, or Post-Mortem Interval (PMI), of skeletonized individuals. To date, bone alterations have been mostly researched in terrestrial settings, such as exposed or buried skeletal remains, but here the scientific literature regarding human bones submerged underwater has been reviewed. It features 20 publications in the last 42 years, of which 9 are reviews, 8 are studies on ancient material and 3 are experimental studies. Future research on analysis of microscopic diagenetic parameters of submerged bones, together with the refinement of the correlation with time of the slightly better known macroscopic underwater alterations, will prove valuable for the estimation of a Post-Mortem Submersion Interval (PMSI) in both forensic and archaeological contexts, because bones have always been and still are regularly recovered underwater. The concurrent estimation of both PMI and PMSI of bones recovered underwater will add vital information to criminal investigations. Diagenetic parameters have been identified in Histological Index, protein content, porosity and crystallinity of bioapatite. They are depicted with the analytic techniques currently available to assess their presence and magnitude, and to relate them to the diagenetic processes of bioerosion, abrasion, and encrustation, but also to the extremes of dissolution or fossilization.

Neural regulation of the function of the gastrointestinal tract (GIT) relies on a delicate balance of the two divisions of its nervous system, namely, the intrinsic and extrinsic divisions. The intrinsic innervation is provided by the enteric nervous system (ENS), whereas the extrinsic innervation includes sympathetic/parasympathetic nerve fibers and extrinsic sensory nerve fibers. In the present study, we used immunofluorescent staining of neurofilament-heavy (NF-H) to reveal the distribution of nerve fibers and their associations with immune cells inside mouse Peyer's patches (PP), an essential part of gut-associated lymphoid tissue (GALT). Our results demonstrate (1) the presence of an extensive meshwork of NF-H-immunoreactive presumptive nerve fibers in all PP compartments including the lymphoid nodules, interfollicular region, follicle-associated epithelium, and subepithelial dome; (2) close associations/contacts of nerve fibers with blood vessels including high endothelial venules, indicating neural control of blood flow and immune cell dynamics inside the PP; (3) close contacts between nerve fibers/endings and B/T cells and various subsets of dendritic cells ( e.g., B220-, B220+, CD4-, CD4+, CD8-, and CD8+). Our novel findings concerning PP innervation and nerve-immune-cell contacts in situ should facilitate our understanding of bi-directional communications between the PNS and GALT. Since the innervation of the gut, including PP, might be important in the pathogenesis and progression of some neurological, infectious, and autoimmune diseases, e.g., prion diseases and inflammatory bowel disease, better knowledge of PNS-immune system interactions in the GALT (including PP) should benefit the development of potential treatments for these diseases via neuroimmune manipulations.

The central nervous system regulates the immune system through the secretion of hormones from the pituitary gland and other endocrine organs, while the peripheral nervous system (PNS) communicates with the immune system through local nerve-immune cell interactions, including sympathetic/parasympathetic (efferent) and sensory (afferent) innervation to lymphoid tissue/organs. However, the precise mechanisms of this bi-directional crosstalk of the PNS and immune system remain mysterious. To study this kind of bi-directional crosstalk, we performed immunofluorescent staining of neurofilament and confocal microscopy to reveal the distribution of nerve fibers and nerve-immune cell associations inside mouse spleen. Our study demonstrates (i) extensive nerve fibers in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, and red pulp; (ii) close associations of nerve fibers with blood vessels (including central arteries, marginal sinuses, penicillar arterioles, and splenic sinuses); (iii) close associations of nerve fibers with various subsets of dendritic cells, macrophages (Mac1+ and F4/80+), and lymphocytes (B cells, T helper cells, and cytotoxic T cells). Our data concerning the extensive splenic innervation and nerve-immune cell communication will enrich our knowledge of the mechanisms through which the PNS affects the cellular- and humoral-mediated immune responses in healthy and infectious/non-infectious states.