Region-specific and age-related differences in astrocytes in the human brain.

Astrocyte heterogeneity and its relation to aging in the normal human brain remain poorly understood. We here analyzed astrocytes in gray and white matter brain tissues obtained from donors ranging in age between the neonatal period to over 100 years. We show that astrocytes are differently distributed with higher density in the white matter. This regional difference in cellular density becomes less prominent with age. Additionally, we confirm the presence of morphologically distinct astrocytes, with gray matter astrocytes being morphologically more complex. Notably, gray matter astrocytes morphologically change with age, while white matter astrocytes remain relatively consistent in morphology. Using regional mass spectrometry-based proteomics, we did, however, identify astrocyte specific proteins with regional differences in abundance, reflecting variation in cellular density or expression level. Importantly, the expression of some astrocyte specific proteins region-dependently decreases with age. Taken together, we provide insights into region- and age-related differences in astrocytes in the human brain.

The Role of Adhesion Molecules and Extracellular Vesicles in an In Vitro Model of the Blood-Brain Barrier for Metastatic Disease.

Metastatic brain disease (MBD) has seen major advances in clinical management, focal radiation therapy approaches and knowledge of biological factors leading to improved prognosis. Extracellular vesicles (EVs) have been found to play a role in tumor cross-talk with the target organ, contributing to the formation of a premetastatic niche. Human lung and breast cancer cell lines were characterized for adhesion molecule expression and used to evaluate their migration ability in an in vitro model. Conditioned culture media and isolated EVs, characterized by super resolution and electron microscopy, were tested to evaluate their pro-apoptotic properties on human umbilical vein endothelial cells (HUVECs) and human cerebral microvascular endothelial cells (HCMEC/D3) by annexin V binding assay. Our data showed a direct correlation between expression of ICAM1, ICAM2, ß3-integrin and α2-integrin and the ability to firmly adhere to the blood-brain barrier (BBB) model, whereas the same molecules were down-regulated at a later step. Extracellular vesicles released by tumor cell lines were shown to be able to induce apoptosis in HUVEC while brain endothelial cells showed to be more resistant.

Spontaneous intracerebral haemorrhage associated with early-onset cerebral amyloid angiopathy and Alzheimer's disease neuropathological changes five decades after cadaveric dura mater graft.

Cerebral amyloid angiopathy (CAA) is a small vessel disease, causing spontaneous intracerebral hemorrhage (ICH) in the elderly. It is strongly associated with Alzheimer disease (AD), as most CAA patients show deposition of Aß-i.e. the basic component of parenchymal Alzheimer amyloid deposits-in the cerebral vessels. Iatrogenic early-onset CAA has been recently identified in patients with a history of traumatic brain injury or other cerebral as well as extra-cerebral lesions that led to neurosurgery or other medical procedures as intravascular embolization by cadaveric dura mater extracts many years before the first ICH event. In those patients, a transmission of Aß seeds from neurosurgical instruments or from cadaveric dura mater exposure was suggested. We report a 51-year-old woman with unremarkable family history who presented abruptly with aphasia and right hemiparesis. A cerebral left lobar haemorrhagic stroke was documented by neuroimaging. Accurate anamnesis revealed a neurosurgical procedure with cadaveric dura mater graft at the age of 2 years for an arachnoid cyst. The neuropathological examination of the cerebral parietal biopsy showed severe amyloid angiopathy in many leptomeningeal and cortical vessels, as well as abundant parenchymal Aß deposits, neurofibrillary tangles and neuropil threads. The mechanism involved in the human-to-human transmission of the Aß proteinopathy remains to be clarified. In our patient the cadaver derived dura used for grafting is a very strong candidate as the source of the transmission. A systematic monitoring of individuals who have had neurosurgical procedures in early life, especially those involving cadaveric dural grafts, is required to determine the ratio of those affected by CAA many years later and unaffected. Moreover, our report confirms that in addition to vascular and parenchymal Aß pathology, neurofibrillary changes indistinguishable from AD may develop in specific conditions with long latency period from the neurosurgical or embolization procedure.

Papaver somniferum in seventeenth century (Italy): archaeotoxicological study on brain and bone samples in patients from a hospital in Milan.

In this paper, we present the results of toxicological analyses of preserved brain tissue and bone samples from the remains of the seventeenth century patients of the Ospedale Maggiore, the main hospital in Milan and one of the most innovative hospitals in Europe from the Renaissance period. Beneath it, the crypt functioned as the burial place for the deceased of the hospital. In this multidisciplinary study of the remains, toxicological analyses in particular were performed with HPLC-MS/MS on different biological samples from nine individuals. Anthropological, paleopathological, histological, radiological examinations and radiocarbon dating were also carried out. As a result, archeotoxicological analyses revealed the presence of codeine, morphine, noscapine and papaverine, derived from Papaver somniferum, a plant present in the hospital pharmacopeia used as a narcotic, analgesic, astringent, coagulant, and antitussive agent. Such analyses have shed light on the pharmacological therapies administered to the patients near the time of death and have implemented our knowledge of medical treatment and drug administration in the 1600's.

Dendritic spine loss in epileptogenic Type II focal cortical dysplasia: Role of enhanced classical complement pathway activation.

Dendritic spines are the postsynaptic sites for most excitatory glutamatergic synapses. We previously demonstrated a severe spine loss and synaptic reorganization in human neocortices presenting Type II focal cortical dysplasia (FCD), a developmental malformation and frequent cause of drug-resistant focal epilepsy. We extend the findings, investigating the potential role of complement components C1q and C3 in synaptic pruning imbalance. Data from Type II FCD were compared with those obtained in focal epilepsies with different etiologies. Neocortical tissues were collected from 20 subjects, mainly adults with a mean age at surgery of 31 years, admitted to epilepsy surgery with a neuropathological diagnosis of: cryptogenic, temporal lobe epilepsy with hippocampal sclerosis, and Type IIa/b FCD. Dendritic spine density quantitation, evaluated in a previous paper using Golgi impregnation, was available in a subgroup. Immunohistochemistry, in situ hybridization, electron microscopy, and organotypic cultures were utilized to study complement/microglial activation patterns. FCD Type II samples presenting dendritic spine loss were characterized by an activation of the classical complement pathway and microglial reactivity. In the same samples, a close relationship between microglial cells and dendritic segments/synapses was found. These features were consistently observed in Type IIb FCD and in 1 of 3 Type IIa cases. In other patient groups and in perilesional areas outside the dysplasia, not presenting spine loss, these features were not observed. In vitro treatment with complement proteins of organotypic slices of cortical tissue with no sign of FCD induced a reduction in dendritic spine density. These data suggest that dysregulation of the complement system plays a role in microglia-mediated spine loss. This mechanism, known to be involved in the removal of redundant synapses during development, is likely reactivated in Type II FCD, particularly in Type IIb; local treatment with anticomplement drugs could in principle modify the course of disease in these patients.

Physical disability in Late Antiquity Milan: slipped capital femoral epiphysis with severe secondary joint disease in the Basilica of San Dionigi.

The paper presents the skeletal remains of an adult male of 30-40 years with bone lesions and deformity on the left hip, recovered during the archaeological excavation below the former Basilica of San Dionigi, dated to Late Antiquity (3rd - 5th century AD) Milan. Biological profile and paleopathological analysis were performed following standard references and the bones underwent radiological examination. Differential diagnosis included congenital anomaly, active rickets, infectious diseases, femoral neck trauma, Legg-Carvé-Perthes disease, Slipped Capital Femoral Epiphysis (SFCE), osteogenesis imperfecta and osteoporosis. While the lesions were highly consistent with Legg-Calvé-Perthes disease, they were typical of SFCE, complicated by an avascular necrosis and secondary osteoarthrosis. The alteration of the femoral head led to a 7.8% leg-length discrepancy, causing gait alteration with partial compensation though increased muscular activity on the right leg, reduced mobility of the joint and potentially hip pain, difficulty in walking and running and even limping. This paper explores a case of physical disability from the Roman era found near a Christian place of worship and represents a rare case of SFCE in the paleopathological record.

GRN-/- iPSC-derived cortical neurons recapitulate the pathological findings of both frontotemporal lobar degeneration and neuronal ceroidolipofuscinosis.

Heterozygous mutations in the gene coding for progranulin (GRN) cause frontotemporal lobar degeneration (FTLD) while homozygous mutations are linked to neuronal ceroidolipofuscinosis (NCL). While both FTLD/NCL pathological hallmarks were mostly investigated in heterozygous GRN+/- brain tissue or induced pluripotent stem cell (iPSC)-derived neurons, data from homozygous GRN-/- condition are scarce, being limited to a postmortem brain tissue from a single case. Indeed, homozygous GRN-/- is an extremely rare condition reported in very few cases. Our aim was to investigate pathological phenotypes associated with FTLD and NCL in iPSC-derived cortical neurons from a GRN-/- patient affected by NCL. iPSCs were generated from peripheral blood of a GRN wt healthy donor and a GRN-/- patient and subsequently differentiated into cortical neurons. Several pathological changes were investigated, by means of immunocytochemical, biochemical and ultrastructural analyses. GRN-/- patient-derived cortical neurons displayed both TDP-43 and phospho-TDP-43 mislocalization, enlarged autofluorescent lysosomes and electron-dense vesicles containing storage material with granular, curvilinear and fingerprints profiles. In addition, different patterns in the expression of TDP-43, caspase 3 and cleaved caspase 3 were observed by biochemical analysis at different time points of cortical differentiation. At variance with previous findings, the present data highlight the existence of both FTLD- and NCL-linked pathological features in GRN-/- iPSC-derived cortical neurons from a NCL patient. They also suggest an evolution in the appearance of these features: firstly, FTLD-related TDP-43 alterations and initial NCL storage materials were detected; afterwards, mainly well-shaped NCL storage materials were present, while some FTLD features were not observed anymore.

Can skin histology be a useful tool for ancestry assessment in forensic settings? Ancestry assessment based on melanin pigment.

Building the biological profile of a deceased person represents a pivotal step in order to achieve the victim's identification. Specifically with regard to ancestry, the melanin distribution pattern in the dermal-epidermal layers has been poorly explored in the forensic field as a potential useful tool. In particular, nothing has been reported about the reliability of such method in bodies in active decay or in advanced state of decomposition. In this study fragments of skin sampled from bodies of known ancestry, both in good and in poor states of preservation, were subjected to histological analysis. We selected 15 subjects, which were divided into three groups: group A (5 white Europeans), group B (5 black Africans) and group C (5 Orientals). A double skin sample was performed on all the bodies, one from the abdomen and the other one from the right forearm. After histological processing and staining with hematoxylin and eosin (H&E) and Masson-Hamperl trichrome technique, the cutaneous melanin distribution pattern was assessed using a semi-quantitative score. The melanin distribution patterns observed both in fresh and in putrefied cadaveric skin were found to be in all cases consistent with the victims' known ancestry. Moreover no differences were observed between abdominal and forearm skin samples and all the histological findings highlighted by H&E were confirmed by the Masson-Hamperl trichrome staining. We demonstrated that the histological analysis aimed at assessing the melanin distribution pattern may be a valuable useful tool in the assessment of ancestry.

DNAJB2-related Charcot-Marie-Tooth disease type 2: Pathomechanism insights and phenotypic spectrum widening.

BACKGROUND AND PURPOSE: Mutations in DNAJB2 are associated with autosomal recessive hereditary motor neuropathies/ Charcot-Marie-Tooth disease type 2 (CMT2). We describe an Italian family with CMT2 due to a homozygous DNAJB2 mutation and provide insight into the pathomechanisms. METHODS: Patients with DNAJB2 mutations were characterized clinically, electrophysiologically and by means of skin biopsy. mRNA and protein levels were studied in lymphoblastoid cells (LCLs) from patients and controls. RESULTS: Three affected siblings were found to carry a homozygous DNAJB2 null mutation segregating with the disease. The disease manifested in the second to third decade of life. Clinical examination showed severe weakness of the thigh muscles and complete loss of movement in the foot and leg muscles. Sensation was reduced in the lower limbs. All patients had severe hearing loss and the proband also had Parkinson's disease (PD). Nerve conduction studies showed an axonal motor and sensory length-dependent polyneuropathy. DNAJB2 expression studies revealed reduced mRNA levels and the absence of the protein in the homozygous subject in both LCLs and skin biopsy. Interestingly, we detected phospho-alpha-synuclein deposits in the proband, as already seen in PD patients, and demonstrated TDP-43 accumulation in patients' skin. CONCLUSIONS: Our results broaden the clinical spectrum of DNAJB2-related neuropathies and provide evidence that DNAJB2 mutations should be taken into account as another causative gene of CMT2 with hearing loss and parkinsonism. The mutation likely acts through a loss-of-function mechanism, leading to toxic protein aggregation such as TDP-43. The associated parkinsonism resembles the classic PD form with the addition of abnormal accumulation of phospho-alpha-synuclein.

Bone tissue preservation in seawater environment: a preliminary comparative analysis of bones with different post-mortem histories through anthropological and radiological perspectives.

Bone taphonomy is a widely investigated topic; however, few data are available concerning marine bone taphonomy, especially on remains recovered from great depths and with short post-mortem intervals. To date, few studies have evaluated the bony changes which occur in seawater compared to samples with different post-mortem histories, and none through a comparative analysis of different approaches. To this purpose, this pilot study aims to examine the influence of seawater on bone preservation compared to other depositional contexts by multiple perspectives. Forty-nine human bone samples (femurs or tibiae) recovered from different environments (sea water, fresh water, outdoor, burial in coffin) were compared by macroscopic, microscopic and bone densitometric approaches. In order to investigate organic and inorganic components, undecalcified and decalcified histology of thin sections was performed. The analyses revealed a well-preserved bone tissue both macroscopically (92%) and microscopically (97% and 95% for undecalcified and decalcified sections). No significant differences were detected from radiological densitometric investigations (BMD = 1.6 g/cm2 ± 0.1), except between old and young individuals (p value < 0.001). Differences were observed for body decomposition and few scavenged samples (3/15). However, even if slight variations were observed, no relation was recorded with the depositional contexts. We found a similar bone preservation in the four environments at the time of recovery, both macroscopically and microscopically, but also with radiological densitometric investigations. Our observations enriched the literature on bone taphonomy, providing data on bone tissue preservation in the early post-mortem period from a multidisciplinary perspective, paving the way for further studies on the topic.

Machine Learning Driven Profiling of Cerebrospinal Fluid Core Biomarkers in Alzheimer's Disease and Other Neurological Disorders.

Amyloid-beta (Aß) 42/40 ratio, tau phosphorylated at threonine-181 (p-tau), and total-tau (t-tau) are considered core biomarkers for the diagnosis of Alzheimer's disease (AD). The use of fully automated biomarker assays has been shown to reduce the intra- and inter-laboratory variability, which is a critical factor when defining cut-off values. The calculation of cut-off values is often influenced by the composition of AD and control groups. Indeed, the clinically defined AD group may include patients affected by other forms of dementia, while the control group is often very heterogeneous due to the inclusion of subjects diagnosed with other neurological diseases (OND). In this context, unsupervised machine learning approaches may overcome these issues providing unbiased cut-off values and data-driven patient stratification according to the sole distribution of biomarkers. In this work, we took advantage of the reproducibility of automated determination of the CSF core AD biomarkers to compare two large cohorts of patients diagnosed with different neurological disorders and enrolled in two centers with established expertise in AD biomarkers. We applied an unsupervised Gaussian mixture model clustering algorithm and found that our large series of patients could be classified in six clusters according to their CSF biomarker profile, some presenting a typical AD-like profile and some a non-AD profile. By considering the frequencies of clinically defined OND and AD subjects in clusters, we subsequently computed cluster-based cut-off values for Aß42/Aß40, p-tau, and t-tau. This approach promises to be useful for large-scale biomarker studies aimed at providing efficient biochemical phenotyping of neurological diseases.

Microglial Heterogeneity and Its Potential Role in Driving Phenotypic Diversity of Alzheimer's Disease.

Alzheimer's disease (AD) is increasingly recognized as a highly heterogeneous disorder occurring under distinct clinical and neuropathological phenotypes. Despite the molecular determinants of such variability not being well defined yet, microglial cells may play a key role in this process by releasing distinct pro- and/or anti-inflammatory cytokines, potentially affecting the expression of the disease. We carried out a neuropathological and biochemical analysis on a series of AD brain samples, gathering evidence about the heterogeneous involvement of microglia in AD. The neuropathological studies showed differences concerning morphology, density and distribution of microglial cells among AD brains. Biochemical investigations showed increased brain levels of IL-4, IL-6, IL-13, CCL17, MMP-7 and CXCL13 in AD in comparison with control subjects. The molecular profiling achieved by measuring the brain levels of 25 inflammatory factors known to be involved in neuroinflammation allowed a stratification of the AD patients in three distinct "neuroinflammatory clusters". These findings strengthen the relevance of neuroinflammation in AD pathogenesis suggesting, in particular, that the differential involvement of neuroinflammatory molecules released by microglial cells during the development of the disease may contribute to modulate the characteristics and the severity of the neuropathological changes, driving-at least in part-the AD phenotypic diversity.

Look before washing and cleaning: A caveat to pathologists and anthropologists.

Partially or totally skeletonized bodies are undoubtedly the most challenging scenario to deal with for forensic pathologists and anthropologists. Indeed, in such cases, being able to figure out the cause and manner of death is often tricky. Human remains require to be washed and cleaned before a thorough assessment of any signs of trauma. However, bones and any fragment of more or less putrefied soft tissues may be home to crucial traces for investigative purposes. They are often located in the context of apparently meaningless dirt and, sometimes they are even invisible to the naked eye. Therefore, their careless cleaning inevitably leads to an unintentional loss of such traces with a negative impact on subsequent investigations. For these reasons before proceeding with cleaning, exhaustive examination and sampling must be carried out. In particular fragments of soft tissues, even if putrefied, are absolutely not to be considered as a hindrance for forensic purposes, since they could still provide valuable information after histological examination. Finally, forward-thinking professionals should think about the possible presence of exogenous micro-traces of forensic concern and collect specimens to be analyzed through in-depth analyses, such as Scanning Electron Microscopy with Energy Dispersive X-ray Spectrometry (SEM/EDX). The present series of cases demonstrates that crucial forensic information can be obtained through the analysis of apparently meaningless residues and even of micro-traces not visible to the naked eye and mixed with trivial dirt.

The frequently dismissed importance of properly sampling skin bruises.

The correct interpretation of skin lesions is a crucial issue in forensic medicine. Many macroscopic and microscopic vital reaction markers and molecules have been studied in the past years in order to estimate the timing of injuries. However, literature lacks information regarding how to perform sampling to obtain a more reliable and accurate response. The present study investigates 25 bruises and analyses the different histological asset in different areas (central or marginal) and the different layers (epidermis, dermis, hypodermis and muscle) of the wound. All wounds were sampled in several areas and analysed under a 200× magnification optical microscope, simply counting the degree of haemorrhaging (i.e hematic infiltration) visible in each optical field (0 = 0%-5%; 1 = 6%-25%; 2 = 26%-50%; 3 = 51%-75%; 4 = 76%-100%). All the injuries presented some kind of infiltration in at least one area, nonetheless only 56% of the wounds were infiltrated in the entirety of the areas. Finally, in 28% the dermal layer did not show any trace of infiltration. Therefore, considering such results it seems that depth or area of sampling of a bruise may largely influence the evaluation of vitality and hence of age of a wound. Though this may seem intuitive, no studies have examined this issue in depth.

Dendritic pathology, spine loss and synaptic reorganization in human cortex from epilepsy patients.

Neuronal dendritic arborizations and dendritic spines are crucial for a normal synaptic transmission and may be critically involved in the pathophysiology of epilepsy. Alterations in dendritic morphology and spine loss mainly in hippocampal neurons have been reported both in epilepsy animal models and in human brain tissues from patients with epilepsy. However, it is still unclear whether these dendritic abnormalities relate to the cause of epilepsy or are generated by seizure recurrence. We investigated fine neuronal structures at the level of dendritic and spine organization using Golgi impregnation, and analysed synaptic networks with immunohistochemical markers of glutamatergic (vGLUT1) and GABAergic (vGAT) axon terminals in human cerebral cortices derived from epilepsy surgery. Specimens were obtained from 28 patients with different neuropathologically defined aetiologies: type Ia and type II focal cortical dysplasia, cryptogenic (no lesion) and temporal lobe epilepsy with hippocampal sclerosis. Autoptic tissues were used for comparison. Three-dimensional reconstructions of Golgi-impregnated neurons revealed severe dendritic reshaping and spine alteration in the core of the type II focal cortical dysplasia. Dysmorphic neurons showed increased dendritic complexity, reduction of dendritic spines and occasional filopodia-like protrusions emerging from the soma. Surprisingly, the intermingled normal-looking pyramidal neurons also showed severe spine loss and simplified dendritic arborization. No changes were observed outside the dysplasia (perilesional tissue) or in neocortical postsurgical tissue obtained in the other patient groups. Immunoreactivities of vGLUT1 and vGAT showed synaptic reorganization in the core of type II dysplasia characterized by the presence of abnormal perisomatic baskets around dysmorphic neurons, in particular those with filopodia-like protrusions, and changes in vGLUT1/vGAT expression. Ultrastructural data in type II dysplasia highlighted the presence of altered neuropil engulfed by glial processes. Our data indicate that the fine morphological aspect of neurons and dendritic spines are normal in epileptogenic neocortex, with the exception of type II dysplastic lesions. The findings suggest that the mechanisms leading to this severe form of cortical malformation interfere with the normal dendritic arborization and synaptic network organization. The data argue against the concept that long-lasting epilepsy and seizure recurrence per se unavoidably produce a dendritic pathology.

Neuropathological Alzheimer's Disease Lesions in Nasu-Hakola Disease with TREM2 Mutation: Atypical Distribution of Neurofibrillary Changes.

Nasu-Hakola disease is a rare autosomal recessive disorder associated to mutations in TREM2 and DAP12 genes, neuropathologically characterized by leukoencephalopathy with axonal spheroids. We report the neuropathologic findings of a 51-year-old female with a homozygous mutation (Q33X) of TREM2 gene. Beside severe cerebral atrophy and hallmarks of Nasu-Hakola disease, significant Alzheimer's disease lesions were present. Neurofibrillary changes showed an atypical topographic distribution being severe at spots in the neocortex while sparing the mesial temporal structures. Our finding suggests that TREM2 genetic defects may favor Alzheimer's disease pathology with neurofibrillary changes not following the hierarchical staging of cortical involvement identified by Braak.

"Aged" autopsy gallstones simulating dry bone context: A morphological, histological and SEM-EDS analysis.

OBJECTIVE: The aim of this paper is to provide information on the morphology and composition of gallstones based on clinical samples in order to assist paleopathologists and bioarchaeologists in recognizing their presence in archaeological contexts. MATERIALS AND METHODS: 270 gallstones were extracted and macerated from autopsies conducted at the Istituto di Medicina Legale in Milan (Italy) in order to simulate a dry bone recovered from archaeological contexts. Morphological, histological, and elemental variation was documented. RESULTS: Gallstones vary in size, shape, color and texture. The cross-sectional surface correlates with chemical composition and is a valuable tool for classification into subcategories of stones. Histological analysis can confirm the classification. Elemental analysis yielded a higher frequency of carbon, calcium and phosphorus. CONCLUSIONS: Although identification of gallstones in archaeological contexts can be challenging, familiarity with morphological, histological, and elemental variation can assist researchers in the field and laboratory. SIGNIFICANCE: Identifying gallstones in archaeological populations will assist researchers in estimating their frequency in the past and the environmental, cultural, and biological conditions leading to their presence. LIMITATIONS: Small sample size derived from a modern and limited autopsy population may minimize the types and degree of variation present in the past. Effects of climate, soil, and taphonomy were not evaluated. SUGGESTIONS FOR FURTHER RESEARCH: Examination of larger samples derived from diverse populations may reveal greater variation or more diagnostic aspects of stones.

Distinguishing Atherosclerotic Calcifications in Dry Bone: Implications for Forensic Identification.

Atherosclerotic calcifications, as calcified atheromatous elements, are markers of cardiovascular disease. However, the literature gives little information regarding their morphological aspect, making their identification very rare in skeletonized cases. In this paper, we document the morphological, histological, and SEM aspects of atherosclerotic plaques collected from unclaimed cemeterial skeletal remains from an identified osteological collection and extracted from well-preserved cadavers autopsied at the medico-legal institute of Milan. Each of the three analyses provided similar results: atherosclerotic calcifications are convex-concave plaques with a stratified structure, a pale-yellow coloration in autopsy cases and yellow to brown when recovered in dry bone. Histologically, undecalcified and decalcified sections showed a stratified aspect formed by superimposed layers. Lastly, the SEM analysis showed a precise view of the stratified structure of the plaques in transverse section. As markers of disease, atherosclerotic calcifications can provide important antemortem information on the deceased that may be compared to antemortem data.